Antimicrobial compositions containing free fatty acids

ABSTRACT

The invention concerns antimicrobial compositions comprising free fatty acids emulsified with membrane lipids or hydrolysed derivatives thereof, and pharmaceutical formulations comprising same. The compositions can be used in the treatment of prophylaxis of microbial infections. They can also regulate the rate of blood clotting rendering them suitable for incorporation in catheter locking solutions and for use in wound care.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.14/623,850, filed Feb. 17, 2015, which is a continuation of U.S. patentapplication Ser. No. 13/510,361, filed Jul. 31, 2012, which is a 371 ofPCT/EP10/67710, filed Nov. 17, 2010, which claims priority to IE2009/00872, filed Nov. 17, 2009, the entirety of which are incorporatedby reference herein.

BACKGROUND OF THE INVENTION

This invention relates to antimicrobial compositions containing freefatty acids and to pharmaceutical formulations containing same.

Free fatty acids are essentially insoluble in water. Their insolubilityand the fact that they are incompatible with many conventionalexcipients have seriously restricted their medicinal use to date. Whilesalts of free fatty acids are soluble in water, they are known to havegreatly reduced antimicrobial effect. Several methods have been used to“solubilise” free fatty acids, including the use of alcohols andsurfactants and derivitisation by re-esterification to formmono-glycerides and/or ethoxylation and propoxylation procedures.

The antimicrobial properties of free fatty acids have been known formany years (Kabara J. et al. Antimicrobial Agents and Chemotherapy, July1972; 2(1): pp 23-28).

Bergson et al. (Antimicrobial Agents and Chemotherapy, November 2001, pp3209-3212), reported that both capric and lauric acid were effective inkilling the yeast Candida albicans.

Sun et al. (Chemico-Biological Interactions 140 (2002), pp 185-198),identified the superior microbicidal properties of caprylic, capric andlauric acid, concluding that lauric was most potent against grampositive bacteria while caprylic was optimal against gram negativeorganisms.

The anti-viral properties of free fatty acids were reported by Halldoret al. (Antimicrobial Agents and Chemotherapy; January 1987, pp 27-31).

WO 03/018049 discloses the antimicrobial activity of milk serumapo-proteins in combination with free fatty acids from milk fat. It isillustrated that the adhesion inhibitory properties of milk extracts areexclusively attributable to the water soluble protein fraction, and thatthe lipid component makes no contribution to this effect.

WO 2009/072097 discloses properties of compositions of free fatty acidssuch as melting point depression and sequestration which affectantimicrobial potency. Also disclosed are emulsification methods used toincorporate blends of free fatty acids in a milk whey protein isolate.

Sprong et al. (Antimicrobial Agents and Chemotherapy, Vol 45, No 4,2001, pp 1298-1301), report microbicidal effects for sphingosine andsphingomyelin and some slight effect from lyso-phosphatidyl ethanolamineand lyso-phosphatidyl choline, but no effect was observed from any ofthe unmodified phospholipids. It is notable that these results arereported for exposure times in excess of 2 hours at 37° C., in contrastto the present invention where microbicidal effects are shown forcombinations of membrane lipids and free fatty acids, for exposure timesof less than 5 minutes.

Jones et al: Journal of Pharmacy and Pharmacology, 2003, Vol 55, No 1.pp 43-52 discloses the use of lecithin in combination with cholesterolas a surface coating to inhibit bio-film formation on medical devices.

It is an object of the invention to provide improved antimicrobialcompositions containing free fatty acids.

SUMMARY OF THE INVENTION

The invention provides an antimicrobial composition comprising:

(a) one or more saturated or unsaturated free fatty acids having from 4to 22, preferably 6 to 18 or 6 to 12, carbon atoms or a pharmaceuticallyacceptable salt or ester thereof; and

(b) one or more membrane lipids or a hydrolysed derivative thereof, asemulsifying agent for the free fatty acid(s) or the salt or esterthereof.

The free fatty acid may be selected from: butyric (C4:0), valeric(C5:0), caproic (C6:0), heptanoic (C7:0), caprylic (C8:0), pelargonic(C9:0), capric (C10:0), undecanoic (C11:0), undecylenic (C11:0), lauric(C12:0), myristic (C14:0), palmitic (C16:0), palmitoleic (C16:1),margaric (C17:0), stearic (C18:0), oleic (C18:1), linoleic (C18:2),linolenic (C18:3), arachidonic (C20:4) and euricic (C22:1).

The free fatty acid is preferably selected from: valeric, caproic,caprylic, pelargonic, capric, undecanoic, undecylenic, lauric, myristic,palmitic, stearic, oleic, linoleic and linolenic acids and mixturesthereof, and pharmaceutically acceptable salts and esters thereof.Particularly preferred are caproic, caprylic, pelargonic, capric,undecylenic and lauric acids, especially caprylic acid.

Where combinations of fatty acids are used including higher meltingpoint entities such as lauric acid, lower melting point entities such ascaprylic or oleic are preferably included to depress the melting pointof the combination to less than normal physiological temperatures.

DETAILED DESCRIPTION OF THE INVENTION

Membrane lipids are ubiquitous components of all cell membranes in theplant and animal kingdoms. They are characteristically made up of one orin most cases two long chain hydrocarbon molecules attached to a highlypolar head group, which is a derivative of either, glycerol-3-phosphate,a long chain amino alcohol (sphingosine), a sugar, or a derivative of asteroid (cholesterol). The properties of each membrane lipid are mainlydictated by the variation in the polar head group. Nearly all areamphoteric in so far as they behave as both acid and base and moreimportantly all are amphipathic, having a water soluble, hydrophilic endat the polar head group and a fat soluble lipophylic end at thehydrocarbon tail.

The physico-chemical properties of membrane lipids are well known and asuitable review of their occurrence and biological properties may befound in Biochemistry, 3rd edition: Mathews, Van Holde & Ahern: ISBN0-8053-3066-6, from which Table 1 has been collated.

TABLE 1 Major classes of membrane lipids. Class of Membrane Lipids PolarHead group Membrane Lipids Lecithin/Phospholipids/ Glycerol Phosphatidicacid Glycerophospholipids Phosphatidylcholine PhosphatidylethanolaminePhosphatidylglycerol phosphatidylinositol PhosphatidylserineSphingolipids & Sphingosine Ceramide glycosphingolipids SphingomyelinGlycoglycerolipids Saccharide Glycolipids GlycosphingolipidsCerebrosides Gangliosides Glycoglycerolipids Mono-galactosyl diglycerideCholesterol Steroids Lanosterol

Of the four major classes of membrane lipids those containing phosphatein the polar head group are the most common. Described asglycerophospholipids, phosphoglycerides or more frequently asphospholipids this group makes up the major portion of all membranelipids in the bacteria, plant and animal kingdoms. All phospholipids arebased on a glycerol backbone with two hydrophobic acyl side chains oncarbons at sn1 and sn2 and a phosphate moiety at sn3. Variations in thephosphate head differentiate six separate types of commonly occurringphospholipids, the simplest being phosphatidic acid and progressing withincreasing complexity through phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidylserine, andphosphatidyl inositol, phosphatidylcholine being the most prevalent inanimals and bacteria.

All six of the above mentioned phospholipids are found in lecithin, amembrane lipid of commerce which is extracted on industrial scale from avariety of sources including but not limited to soya bean, sunflower,canola, palm oil, egg yolk and butterfat. The term ‘lecithin’ isfrequently used synonymously with the major phospholipid componentphosphatidylcholine, which may be purified from crude lecithin, as isusual when rigorous control of quality is required in pharmaceuticalapplications. It is also possible to enzymatically modify lecithin orany of its component phospholipids by, for example, removing one of thehydrocarbon side chains to form ‘lyso-phospholipids’ all of which areequally suitable for use in this invention.

Hydrolysed derivatives of membrane lipids include lyso-phospholipids andlyso-sphingolipids which may be produced enzymatically using pancreaticphospholipases available from Novozyme, Denmark. The process involvespreparation of an aqueous dispersion of phospholipids or sphingolipid,addition of phospholipase enzyme at 2% W/V and incubation at an elevatedtemperature of from about 50° C. to about 60° C. for about 2 hours.Yield of hydrolysed membrane lipid may be up to 70%, and the product maybe separated from the mixture by water partition based on increasedhydrophilicity of the lyso derivative.

The membrane lipids used herein may be extracted from plant or animalsources including oil bearing seeds, animal fat, wool, milk and eggs.

The membrane lipids and derivatives thereof used in the presentinvention are preferably delipidised. As used herein, the term“delipidised” is intended to mean that substantially all of theconjugated extraneous lipid material, such as oil, fat or triglyceridematerial, with which membrane lipids are normally associated in nature,is removed. “Substantially all” in this context is intended to mean thatthe delipidised membrane lipid contains less than 10%, preferably lessthan 5%, more preferably less than 3%, of conjugated extraneous lipidmaterial.

The membrane lipid is preferably selected from one or more ofphospholipids, lecithin, glycerophospholipids, sphingolipids,glycosphingolipids, glycoglycerolipids and cholesterols. Suitablemembrane lipids include lecithin, phosphatidic acid,phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol,phosphatidylinositol, phosphatidylserine, ceramide, sphingomyelin,glycolipids, glycosphingolipids, cerebrosides, gangliosides,glycoglycerolipids, mono-galactosyl diglyceride, lanosterol orcholesterol or any combination thereof. Preferred are lecithin and otherphospholipids selected from phosphatidic acid, phosphatidylcholine,phosphatidylethanolamine, phosphatidylglycerol, phosphatidylinositol andphosphatidylserine and mixtures thereof. Lecithin is particularlypreferred.

In the antimicrobial composition of the invention, a combination of oneor more of caproic, caprylic, pelargonic, capric, undecylenic and lauricacids with one or more phospholipids is particularly preferred,especially caprylic acid with lecithin that is preferably delipidised.

The ratio of free fatty acid or pharmaceutically acceptable salt orester thereof to membrane lipid may be from about 0.25:1 to about 10:1;or from about 0.5:1 to about 10:1, or from about 0.5:1 to about 5.0:1,or from about 1.0:1 to about 2.5:1, or from about 1.25:1 to about 2.5:1,on a weight for weight basis.

Free fatty acids are negatively affected when in contact with bodilyfluids, such as blood, serum or mucus. However, it was unexpectedlyfound that this effect could be ameliorated in the presence of anorganic acid or salt or ester thereof and/or an inorganic acid salt.Thus, the antimicrobial composition of the invention may also compriseone or more pharmaceutically acceptable organic acids or apharmaceutically acceptable salt or ester thereof; and/or one or morepharmaceutically acceptable inorganic acid salts.

The organic acid is preferably selected from acetic, pyruvic, propionic,glycolic, oxalic, lactic, glyceric, tartronic, malic, maleic, ascorbic,fumaric, tartaric, malonic, glutaric, propenoic, cis or trans butenoicand citric acids and mixtures thereof, and pharmaceutically acceptablesalts and esters thereof. The inorganic acid salt is preferably selectedfrom chloride, sulphate and nitrate.

Particularly preferred organic acids are citric and lactic acids and thesodium and potassium salts thereof, especially sodium citrate.

The molar concentration of the organic acid is preferably from about 25mM to about 500 mM, more preferably from about 50 mM to 250 mM, and morepreferably from about 50 mM to 150 mM. The pH of the organic acid saltis preferably between 4.0 and 6.5, preferably between 4.0 and 5.5 orbetween 4.0 and 5.0.

In the antimicrobial composition of the invention, the membrane lipidemulsifies the fatty acid, rendering it water-dispersible. Thus, themembrane lipid-fatty acid combination is an emulsion, preferably anoil-in-water emulsion, although a water-in-oil emulsion is alsopossible.

In a particularly preferred embodiment, the composition of the inventioncomprises an emulsion of 0.5% caprylic acid in 0.4% de-lipidisedlecithin, which is then diluted to 50% of its concentration using 200 mMsodium citrate buffer at pH 4.5, the final concentration being 0.25%caprylic acid emulsified in 0.2% de-lipidised lecithin and dispersed in100 mM sodium citrate at pH 4.5. This composition is referred tohereinafter as the “standard formulation” and may conveniently be usedto demonstrate the antimicrobial effects of the inventive compositions.

The antimicrobial compositions of the invention exhibit a dualantimicrobial effect, in that they exhibit inhibition of microbialadhesion to host cell surfaces and achieve a microbicidal effect throughdissolution of microbial cell membranes.

It has unexpectedly been found that when membrane lipids are separatedfrom conjugated lipid moieties, different membrane lipids exhibitstrikingly different adhesion inhibitory properties on microbial cellsin planktonic culture. While not wishing to be bound by theory, it issuggested that these differences arise from variablehydrophobic/hydrophilic characteristics. Furthermore it has beendiscovered that the same amphipathic variability affects the tenacity ofemulsions formed with individual membrane lipids and that thesedifferences can be exploited to manipulate the rate of release ofemulsified microbicidal free fatty acids, thereby greatly facilitatingthe design of fast or slow acting microbicidal formulations withsuperior utility in medical applications.

As described herein, superior and adjustable adhesion inhibitoryproperties may be obtained using membrane lipids, particularlyphospholipids, collectively described as lecithins, after they have beenextracted and freed of conjugated lipid with which they are normallyassociated in their natural environment. Inhibition of microbialadhesion requires that the lipophylic end of the membrane lipid is freeto orientate with and conjugate to lipid moieties on the microbial cellsurface. This process is impeded if the lipophylic sites are occupied bynon-membrane lipids (triglycerides for example).

A particular utility afforded by the inventive compositions is thediscovery that the different classes and different members of each classof membrane lipids have different release characteristics. The same doseof the same fatty acid in the same amount of different membrane lipidwill deliver the same microbicidal effect over a faster or slower perioddepending on the individual membrane lipid. This peculiar characteristicfacilitates the use of combinations of different membrane lipidemulsions of the same fatty acid to achieve a sustained release effectover relatively useful periods.

A feature of free fatty acids and particularly the short to medium chainsaturated fatty acid, such as caprylic acid, is that they are rapidlyabsorbed through skin and mucosal membranes. Rapid absorption depletesthe dose at the epithelial surface and consequently impairs themicrobicidal effect at that surface. For this reason, membrane lipidsdelivering the most immediate microbicidal effect are not necessarilythe most effective in therapeutic applications. As demonstratedhereinafter in Example 9, certain individual membrane lipids are moretenacious than others, which restricts the availability of theiremulsified free fatty acid, and consequently restricts the rate of itsmicrobicidal effect, which equally restricts its absorption at theepithelial surface. It should be noted, however, that the overallmicrobicidal effect (log numbers of microorganisms killed) is notaffected.

It has also unexpectedly been found that the antimicrobial compositionsof the invention can be used to regulate the rate of blood clotting. Byvarying the ratio of free fatty acid to membrane lipid and/or byincorporation of pharmaceutically acceptable salts of organic orinorganic acids, or oligosaccharides or other polymers, the formulationswill either catalyse the rate of blood clotting or suppress italtogether. This property gives great advantage in use as a bloodcontact antimicrobial agent.

As disclosed herein, an aqueous dispersion of de-lipidised membranelipid added to fresh sheep blood at a volume of 20% will accelerate thenormal rate of blood clotting, reducing the time to clot from 6 minutesto less than one minute. Addition of a fatty acid, such as caprylicacid, by emulsification up to a ratio of about 1.0-1.3 times the weightof de-lipidised membrane lipid, such as lecithin, will further reducethe time to clot formation, but thereafter as the ratio of fatty acidincreases, the time to clot formation is increased and ananti-coagulation effect is observed when the weight of emulsified fattyacid exceeds about 1.0-1.3 times the weight of de-lipidised membranelipid. A skilled person will appreciate that the blood regulatory effectwill depend not only on the ratio of free fatty acid to de-lipidisedmembrane lipid, but also on the nature of the membrane lipid and freefatty acid used.

The presence of an organic salt in the inventive composition, as mightbe required for amplification of microbicidal effect, will also destroythe pro-coagulation effect, and as illustrated herein the use of aviscosity-enhancing agent, such as dextran, will extend theanti-coagulation effects to the point where they are comparable with asolution of heparin.

In a further embodiment, an emulsion of free fatty acid not exceedingabout 1.0-1.3 times the weight of de-lipidised membrane lipid may beused to enhance the rate of blood clotting and exert an antimicrobialeffect at the site of bleeding, while an emulsion of free fatty acidexceeding about 1.0-1.3 times the weight of de-lipidised membrane lipidwith or without added salts of organic acids may be used to inhibitblood clot formation and exert a microbicidal effect at the site ofbleeding.

The compositions of the invention containing membrane lipid emulsifiedfree fatty acids exert vastly superior dual antimicrobial effects: bothinhibition of adhesion and microbicidal effect. One of the significantadvantages provided by dual antimicrobial effect is that the adhesioninhibitory properties prevail long after the microbicidal pay-load hasbeen exhausted. Most microbicides are chemically reactive with thetarget organism and most microbicidal reactions are irreversible underphysiological conditions; a fixed dose of a microbicide therefore has alimited reactive capability. In combination with an adhesion inhibitorysubstance however, the adhesion inhibitory properties persist after themicrobicide has been depleted and additional protection is affordedagainst any residual viable pathogens.

In addition to superior and adjustable inhibition of adhesion combinedwith superior microbicidal effect and intrinsic blood-clottingregulatory effects, the compositions of the invention are compatiblewith systemic administration (blood contact), in contrast to milkprotein compositions disclosed in WO 03/018049, and WO 2009/072097.Membrane lipids and free fatty acids are natural metabolites in thehuman and animal body, their antimicrobial effects are concentrationdependent and when they enter the systemic circulation they are rapidlydiluted, metabolized and excreted as natural metabolites.

The use of membrane lipids in combination with an antimicrobial agent iscounter-intuitive, as most conventional antimicrobial agents areinactivated by lecithin and related membrane lipids; lecithin is listedas an approved antiseptic neutralizing agent for use in EuropeanStandard EN 1499 and 1500 testing for evaluating the microbicidal effectof hand soaps and gels.

The compositions of the invention may be used in the treatment orprophylaxis of microbial infections in humans or animals. Due to theregulatory effect on blood-clotting, the compositions may be used in,for example, catheter locking solutions and in wound care.

The invention also provides a pharmaceutical formulation comprising acomposition according to the invention and a pharmaceutically acceptablecarrier, diluent or excipient therefor. The composition may be presentin the pharmaceutical formulation in an amount of from about 0.1% toabout 25% (w/v); or from about 0.1% to about 10% (w/v); or from about0.1% to about 1.0% (w/v).

The pharmaceutical formulation may be in any form suitable foradministration to a human or animal, including for example, formssuitable for oral, topical, enteral, parenteral or mucosaladministration.

The formulation of the invention can be used as a topical antimicrobialagent for prevention and treatment of infections of the skin, hair,nails and external membranes of the body orifices.

The inventive composition or formulation may be used in the constructionof a liquid soap or hand gel for elimination of asymptomatic carriage ofpotentially pathogenic bacteria such as methicilin resistantStaphylococcus aureus and other species commonly associated withnosocomial or hospital acquired infections. It may be used as a gel fortreatment of acne. It may be used in the form of an ointment fortreatment of dermatophytic fungal infections of the hair folliclesincluding that caused by Trychophyton species commonly known asringworm. It may be constructed in the form of a spray for delivery tothe skin as a treatment for infections caused by the enveloped virusesincluding Herpes varieties causing cold sores and shingles. It may beprepared in the form of liquid drops for treatment of infections of theeye and ear. It may be prepared in a variety of pharmaceuticallyacceptable delivery systems for treatment of infections of the mucosaincluding the mucosal surfaces of the nose, mouth, throat, bronchioleand lungs and the gastro-intestinal tract and genitalia. It may forexample be prepared in the form of a toothpaste and mouthwash tofacilitate improved dental hygiene and eliminate the burden of organismscausing dental caries, gum disease, aphthous ulcer and halitosis. It maybe prepared in a form suitable as a saliva supplement for alleviation ofthe symptoms of xerostomia. It may be prepared as a spray forde-colonisation of the mouth, throat and nasal membranes, particularlyfor eradication of asymptomatic carriage of antibiotic resistantspecies. It may be prepared in the form of a gel for the prevention andtreatment of microbial infections of the genitalia caused by a varietyof bacteria, yeast and viruses including those known to be the causativeagents of Candidiasis, non-specific bacterial vaginosis, the herpesvirus and HIV. It may be prepared in the form of an enema for preventionand treatment of infections of the bowel and lower intestinal tractincluding those caused by anaerobic Clostridium species andDesulfovibrio medically known as pseudomembraneous colitis andpouchitis.

Membrane lipid emulsified free fatty acids may be used to prepare foodsthat exert an antimicrobial effect in the gastro-intestinal tract,serving to protect against enteric pathogens such as Helicobacter, E.coli, Salmonella, Campylobacter, Clostridium species, Protozoa andenveloped viruses.

A suitable food carrier for membrane lipid emulsified free fatty acidsis dairy milk, preferably fat free dairy milk and more preferably skimmilk powder such as Marvel, available commercially from PremierInternational Foods (UK) Ltd, Spalding, Lincolnshire, England.

Separate emulsions of, for example, caprylic, capric and lauric acidsmay be prepared as 5.0% W/V emulsions in 4.0% W/V de-lipidised lecithinas described in the methods. The individual emulsions may be combined ina ratio of 1:1:1 or any other suitable ratio such as 1:2:3. Membranelipid emulsions of other fatty acids such as butyric and or emulsions ofessential oils such as peppermint or vanilla may be added to impartflavour and improve taste.

Marvel skim milk powder may be re-constituted by adding the specifiedamount to potable water (4 heaped teaspoons to one pint). To this anamount from about 1% W/V to 15% W/V of a selected ratio of membranelipid emulsified free fatty acids may be added and mixed by stirring.Preferably the amount of emulsified fatty acid will be from about 1% W/Vto 10% W/V and more preferably about 5% W/V. It will be clear to askilled person that skim milk powder may be re-constituted in less thanthe optimal volume of water for use as milk, in which case aconcentrated dairy cream is formed to which amounts of emulsified fattyacids may be added.

Alternatively, a dairy whey protein isolate may be used as a suitablefood carrier: Provon 190 from Glanbia PLC is a suitable whey proteinisolate. The whey protein isolate may be re-hydrated in potable water inan amount of from about 10% W/V to 20% W/V, preferably about 15% W/V.Once fully hydrated, an amount of membrane lipid emulsified free fattyacid or blend thereof may be added and mixed by stirring. When wheyprotein isolate is used as a carrier, the amount of added membrane lipidemulsified free fatty acid may be from about 1% W/V to 20% W/V,preferably from about 5% W/V to 15% W/V and more preferably about 10%W/V.

The products of the invention have utility as blood contactantimicrobial agents where their combined blood clotting oranti-clotting capability is combined with their antimicrobial effects.Such uses include surgical irrigation, wound care, catheter lockingsolutions, the coating of catheters and other tubular surgical devicesfor insertion into a bodily orifice or cavity, and in food safety.

A significant advantage of the products of the invention is that veryshort exposure times are required to achieve an antimicrobial effect,generally less than 1 hour or less than 30 minutes or less than 10minutes.

As used herein and in conventional use, the term ‘antimicrobial’ refersto any substance, component or composition of components which exhibitan antagonistic effect towards protozoa, gram positive and negativebacteria (both aerobes and anaerobes), yeast, fungi, mycoplasma and/orviruses and in particular those microbial species that are capable ofcausing disease in humans and animals.

Infectious disease arises either from ingress of pathogenic microbialspecies (microorganisms) from the external environment or as a result ofaberrant growth of microorganisms that are normally present in thenatural micro-flora of the skin, hair, and mucosal membranes of the eye,nose, mouth, gastro-intestinal tract and the genitalia.

Whether exogenous or endogenous it is widely understood among healthcareprofessionals that the first stage of microbial pathogenesis involvesadhesion of the microorganism to the surface of human or animal tissue.Once adhered, colonization takes place by way of proliferation andfurther adherence after which toxin production inflammation anddestruction of host tissue give rise to the classical symptoms ofinfectious disease. It is also widely accepted that if adhesion can beprevented, initiation of the pathogenic process can be inhibited andmany infectious diseases could be prevented or at least limited in thescope of their proliferation.

The novel properties of membrane lipid emulsified free fatty acidspresent wide ranging utility in human and animal healthcare. In bloodcontact applications these include surgical irrigation fluids,haemostatic antimicrobials in wound care, catheter coatings and catheterlocking solutions for prevention of catheter related bacteraemia; inmucosal healthcare they can be used to replicate the naturalantimicrobial mechanism of healthy mucus; in topical skin care they canbe used to amplify the natural antimicrobial defenses of the skin; infood safety because they are natural metabolites they can be applieddirectly to a food surface to eradicate food borne pathogens; and asmedical foods they can be used to supplement a normal diet to prevent ortreat infections of the gastro-intestinal tract.

Surgical Irrigation and Wound Care

During surgical intervention it is common practice to employ a varietyof techniques to minimize the ingress of potentially pathogenicbacteria, yeasts, moulds and viruses. A common and growing problem isthe potential infection of an open wound by antibiotic resistantbacteria such as Methicillin Resistant Staphylococcus Aureus (MRSA), andmany others including but not limited to Enterococcus species,Pseudomonas, and the yeast Candia albicans. In many cases open woundsare irrigated to clear them of loose tissue, blood and other body fluidsand in many cases a solution of sterile saline is currently in use,although this offers no antimicrobial effect. The use of surgicalirrigating fluids containing antibiotics is not recommended simplybecause these have the potential to generate further antibioticresistant species. Equally, many conventional antiseptic agents areunsuitable and potentially toxic in direct systemic contact with an openwound. There is therefore a great need for a safe and effectiveantimicrobial irrigating fluid, preferably one which has the additionaloptional properties of inhibiting blood clotting during microsurgeryand/or a companion product which can promote blood clotting and healingafter surgery. Compositions based on optionally de-lipidised membranelipids combined with free fatty acids and/or derivatives thereof asdescribed herein provide such utility. Also in trauma care afteraccidental injury or during military operations, wounds are frequentlyjagged, dirty and possibly hemorrhaging profusely. There is a great needfor emergency intervention with antimicrobial products that promoteblood clotting and which can be applied safely to an open wound.

Prevention of Catheter Related Infection

In simplistic terms a catheter is a tube inserted through the skin, intoan artery or vein or through a natural orifice for the purpose ofdraining body fluids, or for administration of drugs or for the purposeof monitoring disease or manipulation of surgical instruments. Somecatheters remain in place for relatively long periods of time, and inmany cases these are susceptible to microbial contamination causingbio-film on the inside and outside surfaces and potentially leading todisseminated infection of the patient.

The most common catheter is a urinary tract tube with a collectingvessel to drain urine from the bladder; these are recognized as beingsignificant sources of hospital acquired infection. A more elaborate andcomplicated system is a central venous catheter (CVC) inserted eitherthrough the jugular vein in the neck and threaded through to thesuperior vena cava at the heart or inserted through a peripheral veinand threaded through to the same vein draining into the heart. CVCs aredesigned to be left in place for extended periods of up to 90 days ormore and are intended for long term repetitive infusion of drugs and/ornutritional formulations; in such applications they are routinely openedfor administration and closed again for intermittent periods duringwhich fluid in the lumen of the catheter is static and susceptible tomicrobial growth if contaminated. Accidental contamination of CVCs isrelatively common and frequently gives rise to catheter related bloodstream infections which are potentially fatal.

Blood enters the lumen of a CVC during routine medical procedures, whereit clings to the inside and may clot blocking the lumen. A blocked CVClumen necessitates catheter replacement under surgery which adds greatlyto time, cost and patient mortality.

A catheter locking solution (CLS) is a volume of fluid sufficient tofill the lumen of the catheter between medical procedures. An optimalCLS will exert an antimicrobial and anti-coagulation effect. Currentlythere are a number of proprietary CLS formulations on the market thatare designed to achieve both prevention of blood clotting and anantimicrobicidal effect. The selection of active ingredients toconstruct these CLS formulations is constrained by the risk ofaccidental intra-venous infusion. The void volume of a CVC may be asmuch as 3.0 ml or more. In the event that a medical administrator forgotthat a locking solution had been inserted and accidentally added asecond dose, the first void volume would be infused directly into theblood stream where it could have serious toxicological implications forthe patient.

The formulations herein may be in the form of catheter lockingsolutions, which can achieve optimal microbicidal effect in less than 1hour and have anti-clotting properties similar to heparin without theuse of that material in the formulation. The catheter locking solutionspreferably have a viscosity approximating to that of whole blood tominimize the potential for dilution and mixing at the catheter tip. Thedesired viscosity can be achieved using viscosity-enhancing agents.

Viscosity-enhancing agents routinely used in pharmaceutical preparationsinclude a wide range of hydrogels of natural or synthetic origin.Included among these are derivatives of cellulose such as carboxymethylcellulose and hydroxyethyl cellulose. Other natural polymers of plantorigin include dextran, alginate, pectin, guar gum and acacia gum;synthetic polymers include a range of carbomers (acrylates/C10-30 alkylacrylate cross polymers) and Poloxamers (triblock copolymers ofpolyoxyethylene and polyoxypropylene). For reasons of residual toxicityand metabolic clearance, few of these are approved for routine systemicuse in human medicine. The Poloxamer, Pluronic F68 has been used inparenteral nutrition formulations, but the naturally occurring dextranhas achieved more extensive use in surgery.

In the formulations of the invention, dextran is the preferredviscosity-enhancing agent. Dextran is a naturally occurringpolysaccharide. A complex branched glucan of glucose monomers, it isproduced by many bacteria including Leuconstoc species, and it has beenused as an irrigant and anti-clotting agent in micro-surgery. Dextran isavailable commercially in a range of molecular weights ranging from 10Kilo daltons (Kd) to 150 Kd. In the invention, 20 Kd to 60 Kd dextran ispreferred. More preferably, 40 Kd dextran is used to adjust theviscosity of a membrane lipid/free fatty acid-based catheter lockingsolution to approximate to that of whole blood: 3.6-6.5 cP.

When 40 Kd dextran is used as viscosity-enhancing agent, itsconcentration in formulations should preferably be from about 5% to 50%on a weight by volume basis, more preferably from about 10% to 40%, andmore preferably still from about 15% to 30% weight by volume based onthe entire formulation.

Table 2 below provides a summary of the more common proprietary CLSformulations currently in use:

TABLE 2 Proprietary CLS Formulations currently in use or underdevelopment Proprietary Brand Known Active Ingredients Reported EfficacyDuralock 47.6% Tri-sodium citrate Anti-coagulant, MedComp, microbistaticnot Philadelphia USA microbicidal Zuragen 7% sodium citrateAnti-coagulant Ash Access 0.05% Methylene Blue Microbicidal overTechnologies 0.15% Methyl Parabens 12 hours Lafayette, Indiana, USA0.015% Propyl Parabens Taurolock 1.35% Taurolidine Anti-coagulantTauropharm AG, 4% sodium citrate Microbicidal over Waldebuttelbrunn 12hours Germany Canusal 0.9% saline Anti-coagulant Wockhardt UK LTD 200I.U. heparin/ml Not microbicidal Wrexham Wales

Most of the lock solutions in use currently have relatively lowmicrobicidal effect: reduction in microbial viability of 3-4 logs inmore than one hour exposure. As disclosed herein, a CLS based onmembrane lipid emulsified free fatty acids exhibits superiorantimicrobial effect eradicating greater than 6 logs in less than 6minutes. Optimal anti-coagulation effects are achieved by modifying theratio of membrane lipid and free fatty acid and the optional use of aviscosity-enhancing agent eliminates migration of blood into thecatheter tip. Greater safety in the event of accidental double lockingis assured by the fact that the components used in this invention arenatural metabolites.

Antimicrobial Surface Coating

The compositions of this invention can be used to create dual actionanti-adhesion and antimicrobial surface coatings by trapping a film offree fatty acid, on any animate or inanimate surface, including but notlimited to skin, plastic, rubber, metal or glass, wherein they exert amicrobicidal effect at the surface in addition to repelling adhesion ofmicrobial species.

Active antimicrobial surface coatings have particular application inhealthcare, for prevention of bio-film formation on medical instrumentsand on catheter surfaces, and also as a surface coating for workstations, procedural trays and all patient contact surfaces, includingthe hands of healthcare workers. Similarly, active antimicrobial surfacecoatings have extensive application in the food industry where they canbe applied to food preparation and packaging surfaces to minimizecarriage of food borne pathogens such as Salmonella, Campylobacter,Listeria and E. coli and as demonstrated herein, they can also beapplied to the surface of food products, particularly post-slaughtermeat, to eradicate these pathogens at source.

Conventional surface antisepsis is achieved using an antiseptic wipewhich may contain triclosan, chlorhexidine, quaternary ammoniumcompounds or a concentrated solution of alcohol. However, the residualtoxicity of many conventional antiseptics limits their use on food andanimate surfaces.

When used in surface coating applications, the composition of theinvention may be applied in a suitable pharmaceutically acceptabledelivery system, with or without other excipients. Pharmaceuticallyacceptable delivery systems include, but are not limited to, organicsolvents designed to evaporate on application leaving a dry residue,liquids, creams, gels, pastes, ointments, powders or sprays and includecombinations of these with insoluble materials such as fibrous wipes.

Mucosal Fortification:

The mucus membranes of humans and animals are characteristically moistsurfaces at the interface of natural body orifices, and the lining ofthe gastro-intestinal tract and the genitalia, they include the eye,nose, inner ear, mouth, naso-pharyngeal surfaces, trachea, bronchioli,esophagus, stomach, large and small intestines, rectum, vagina andexternal labia, the glans and the lining of the urinary tract. Inaddition to these anatomically related surfaces that are common tohumans and animals, there are mucosal structures that are unique toparticular species such as the guttural pouch in equids.

Mucus membranes are covered by a layer of mucus, a viscous hydrocolloidcomprised mainly of mucins, a group of heavily glycosylated highmolecular weight proteins which act as a matrix within which many otherbiologically active materials are dispersed including secretoryantibodies and components of the innate immune system such as histatinsand statherins in saliva. In addition to hydration and lubrication,mucus is essential for prevention of ingress of potential microbialpathogens and for prevention of adhesion and colonization of the mucusmembranes themselves.

Impairment of mucosal secretions and debilitation of the integrity ofthe mucus itself may arise as a consequence of disease, or as a sideeffect of medical and/or pharmaceutical intervention or as a consequenceof life-style. Where this happens, those afflicted suffer from recurringmucosal infection including but not limited to dental caries,gum-disease, oral thrush, yeast vaginitis, bacterial vaginosis,enteritis and infectious colitis. The complexity of mucus has defied allattempts to construct exact replicas which may be used in replacementtherapy. There are a number of commercial substitutes, which aredesigned to relieve the pre-dominant physical symptoms of oral dryness.Most of these are based on hydro-gels such as carboxymethyl celluloseand a composition of salts to affect buffering and re-mineralisation.One is based on pig-gut mucin (Saliva Orthana, AS Pharma, Eastbourne,UK), but none approximate to the natural adhesion inhibitory aspects ofsaliva and mucus in general. The combined adhesion inhibitory andmicrobicidal properties of the inventive formulations, particularlytailored release formulations as disclosed herein, offer superiormimicry of the antimicrobial properties of natural mucus.

Topical Disinfection and Skin Care

The skin, hair and nails of mammals, being the external body surfacesare subject to constant environmental contamination. Healthy mammalianskin has intrinsic antimicrobial properties based on naturally occurringfree fatty acids in the skin, and these may be advantageously fortifiedby topical application of delipidised membrane lipid-emulsified freefatty acids as disclosed herein.

Bacteria causing skin infection include, but are not limited to,Staphylococcus aureus, Streptococus pyogenes and Pseudomonasareruginosa, causing Impetigo, Folliculitis, Erysipilis, Cellulitis andNecrotising Fasciitis. Propionibacteriium acne is the causative agent ofjuvenile acne. Fungal skin infections are caused mainly by Trichophyton,Microsporum and Epidermophyton species and the diseases are knowncollectively as Tinnea (pedis, cruris, capitis, corporis and unguinium)which include ringworm and nail infections. Yeast including Candidaalbicans cause Intertrigo and Paronychia; Malassezia furfur causesTinnea versicolor (seborrheic dermatitis and infectious dandruff) and itis also a common ailment in dogs' ears. Enveloped viral infectionsinclude Herpes Simplex Type 1 affecting orofacial regions and type 2affecting the genital regions. Herpes zoster causes shingles and thepoxvirus causes Molluscum contagiosum. Superficial asymptomatic carriageof HIV, SARS, Hepatitis, Swine flu′, bird flu′ and many other zoonoticviral infections may also be eliminated using the compositions herein.

There is significant public concern about the increasing incidence ofhospital acquired infection (HAI). HAI include infections caused byantibiotic resistant bacteria such as Methicilin ResistantStaphylococcus aureus (MRSA), and Vancomycin Resistant Enterococci (VRE)and other multi-drug resistant species such as Clostridium difficile andother less well known opportunistic pathogens including Pseudomonas andCandida.

It is well known that hand antisepsis is critical in preventingcross-contamination between patients and patient care staff. Mostpatient care establishments routinely use an alcohol gel for handantisepsis. Alcohol is an immediate and potent microbicidal agent. Itevaporates within seconds however, leaving no persistent effect toprotect against accidental contamination that might happen immediatelyafter evaporation. Membrane lipid emulsions of free fatty acids may betailored in a manner that provides a sustained release of microbicidaleffect and when formulated with alcohol, the effect is both immediateand persistent.

Food Safety

It is well known that many food animals harbour food borne pathogens,primarily in their gut, but also on their skin from contamination withfaeces. Conventional antiseptics and antibiotics are not suitable forliberal topical or systemic application to animals before slaughter, orto their meat after slaughter. The individual components that make upthe products of this invention, however, have the advantage of beingused routinely in parenteral nutrition; in separate constructs thereforethey have been deemed to be non-toxic, and their suitability for usetopically on fresh meat is disclosed herein.

Food borne pathogens include Salmonella, Escherichia, Campylobacter andClostridium species. The efficacy of the products of this invention invitro is demonstrated in the Examples. Methods of eradication of foodborne pathogens at primary processing of meat include the proposed useof carcass washing with a suitable antimicrobial agent. Other methods ofapplication that are relevant to food safety include the use of membranelipid-emulsified free fatty acids as surface coatings for food packagingusing procedures similar to those described for application ofantimicrobial surface coatings to medical devices.

Methods and Materials:

Emulsification Procedures

Free fatty acids greater than C6 are insoluble in aqueous media.Membrane lipids are equally insoluble in water but may be dispersedtherein, and under appropriate conditions combinations of free fattyacids and membrane lipid may be induced to form emulsions in water.These are either oil-in-water or water-in-oil emulsions, the latterhaving the higher relative concentration of fatty acid by weight.

A water-in-oil (free fatty acid) emulsion may be prepared by dissolvinge.g. about 1 gram of membrane lipid, such as optionally delipidisedlecithin, in about 2 grams of free fatty acid. Stir for about 10 minutesand then add about 2 grams of sterile distilled water, agitatevigorously by shaking for 30 seconds and allow to hydrate for 30minutes. When fully hydrated the emulsion has a thick creamy consistencywhich may be further diluted with up to 20 ml of water, added in 5 mlaliquots with intermediate agitation by shaking. An excess of wateradded to this emulsion will cause it to break and invert in part or inwhole and become unsuitable for further processing.

Oil-in-water emulsions are better suited to high water contentapplications such as wound dressings, catheter locking solutions andsurface disinfection, as described hereinafter. Methods of preparingoil-in-water emulsions are well known to those skilled in the art andare disclosed in, for example, WO2009/072097.

A suitable example of an oil-in-water emulsion of free fatty acid inmembrane lipid may be prepared by suspending a suitable quantity ofmembrane lipid, such as optionally delipidised lecithin, in steriledistilled water, and stirring this at room temperature for about 30minutes with a magnetic stirrer to ensure even dispersion. Using asuitable dispersing device, the desire amount of free fatty acid, suchas caproic, or caprylic, or pelargonic acid, is slowly added whilevigorously agitating the volume of dispersed membrane lipid. The amountof membrane lipid (e.g. lecithin) is conveniently 0.4 g in 100 ml water,to which 0.5 g of free fatty acid can be added using e.g. a 5-ml syringeor pipette. A laboratory homogenizer such as an Ultra-Turax Model T18(IKA Works, Wilmington, N.C. 28405, USA) fitted with an S18N-19Gdispersing tool, operating at 6,000 to 8,000 RPM, is a preferred methodof agitation. The resulting emulsion is a thin white liquid suspensionof free fatty acid, which is stable and can be diluted many-fold withoutde-stabilising the emulsion.

A similar procedure may be used to prepare an oil-in-water emulsion of,for example, capric acid or undecylenic acid, provided the constituentsare first equilibrated at 37° C., or above their melting point. Onceemulsified, the emulsion remains stable on cooling below the meltingpoint of the incorporated free fatty acid and may be diluted further foruse in formulation.

A membrane lipid emulsion of lauric acid may be prepared in a similarmanner provided all constituents are first equilibrated at 50° C. orabove the melting point of lauric acid, and again this emulsion willremain stable on cooling and in further dilution for use in formulation.

Free fatty acids have limited microbicidal effect at temperatures belowtheir individual melting point. This fact has little significance forthose acids with melting points below normal physiological temperatures(37° C.), except in situations where such emulsions may be required toexhibit an effect in hypothermic conditions.

Blends of high and low melting point free fatty acids have depressedmelting points due to the solvent effect of the lower melting pointconstituent. A combination of lauric acid with a melting point of 44° C.and caproic acid with a melting point of −3.4° C. in a 50:50 ratio, willexhibit a combined melting point of 26° C., and the blend may beemulsified as described above at temperatures above 26° C.

Higher melting point free fatty acids may also be dissolved in lowmelting point blends of essential oils or other neutral oils to achievea depression of melting point suitable for antimicrobial use underphysiological conditions. Essential oils are hydrophobic constituentsextracted from many different plants by distillation or solventextraction. They are widely used commercially as perfumes and in fringemedical practice such as aromatherapy. Most notable examples include oilof clove, orange, lemon, lavender, juniper, and rose. Many of these areknown to exhibit microbicidal effect in their own right; oil of lemonbalm for example is recognized as being an effective viricidal agent andwhen used as a solvent for higher melting point fatty acids such aslauric acid, the combined microbicidal and viricidal properties provideexpanded utility in medical applications.

Oil of lemon balm will dissolve up to 40% by weight of lauric acid at20° C. and the blend may be emulsified in membrane lipid such aslecithin, as described above.

Oil-in-water emulsions of free fatty acids or blends thereof may beprepared in concentrations of optionally de-lipidised membrane lipidranging from about 0.1% to 10% and emulsified free fatty acid loadingsof from 0.1 to 10%. The loading may conveniently be 4% membrane lipidsuch as lecithin, and 5% free fatty acid or blend thereof. Morepreferably still a loading of 0.4% optionally de-lipidised lecithin and0.5% free fatty acid or blend thereof is used, which may be dilutedfurther for use in formulation.

The microbicidal efficacy of any membrane lipid emulsion of free fattyacid according to the invention is amplified by increasing surface areaof the emulsified droplet; relative surface area is increased bydecreasing the droplet size. Droplet size is a function of the amount ofenergy imparted by the dispersing tool during the emulsificationprocedure. Generally, finer droplets may be produced using higherhomogenization speeds and the use of an ultrasonic probe adjacent to theemulsification head will also facilitate smaller droplets and increasedsurface area.

De-Lipidisation of Membrane Lipids

De-lipidisation can be achieved by suspending the membrane lipid orhydrolysed derivative thereof in a polar solvent, such as an alcohol orketone, at a concentration of about 10% w/v and stirring for about 30minutes during which time extraneous lipid is dissolved. Membrane lipidsare insoluble in polar solvents and will readily settle out afterstirring allowing the polar solvent with its dissolved lipid to bedecanted. Residual solvent is allowed to evaporate in e.g. a fume hood.Suitable solvents include acetone and ethanol.

Adhesion Assay:

A suitable model of microbial adhesion and inhibition of adhesion isprovided by the interaction between the yeast Candida albicans and humanBuccal Epithelial Cells freshly harvested from the inside the cheek.

In the assay procedure, a standardized population of fresh latelog-phase Candida are exposed to a variable concentration of testsubstance for 10 minutes, then combined with a fixed ratio of buccalepithelial cells (BECs) for 60 minutes, during which time yeast willadhere to the BECs to a greater or lesser degree depending on thepotency of the inhibitory substance used in pre-treatment. After theadhesion period, the combined yeast and BECs are diluted by a factor of2× in sterile buffer and agitated briefly (5 seconds) on a laboratoryvortex. Wet mount samples of the mix were examined under a microscopeusing 400× magnification. Yeast cells adhering to BECs are clearlyvisible and enumeration of these is facilitated using dark fieldconditions; the use of a Neubauer hemocytometer slide facilitatescounting. In total 100 BECs are evaluated and the total number of yeastadhering to these is used as the sample count. The control is equivalentto 100% adhesion and reduction in this number in response to test itemor protein blank is reported as percent inhibition of adhesion.

Buccal epithelial cells are harvested from the inner cheek mucosa ofvolunteers by rubbing a sterile tongue depressor in a circular fashionand rinsing the collected cells into 5 ml of sterile phosphate bufferedsaline (PBS). BECs should preferably be collected in the morning beforeeating or brushing teeth. Donations from 4-5 volunteers are needed toachieve five assay points.

BECs are pooled and counted using direct microscopic count with ahemocytometer slide; a count of 500/ml (for example) may be achieved.The pooled sample is centrifuged at 1,500 RPM for 3 minutes, andre-suspended in a volume of sterile PBS, calculated to achieve aconcentration of 500 BECs per ml. This concentrate was divided into 2.5ml aliquots in 10 ml centrifuge tubes and centrifuged again at 1,500 RPMfor 3 minutes. The supernatant is discarded and the cell pellets held onice pending their use in the rest of the assay.

The yeast used in these assays is a fresh clinical isolate of Candidaalbicans; ATCC 10231 may be used as an alternative, but all culturecollection ‘Type’ strains have lost some virulence and do not adhere aswell as fresh isolates.

Yeast cells are maintained on yeast extract peptone dextrose (YEPD)agar, and a loop-full of a pure culture is used to inoculate a 250 mlErlenmeyer flask containing 100 ml of sterile YEPD broth. The inoculatedflasks are incubated overnight (10-14 hours) at 37° C. in an orbitalincubator at 100 RPM. YEPD is: 2% W/V glucose, 1% W/V Yeast Extract, 1%W/V bacteriological peptone, Agar where required at 1-2% W/V, all fromOxoid, UK.

The late log-phase cells are counted with a hemocytometer slide and theconcentration adjusted to 100× the test concentration of BECs (40,000/mlin these examples), using 50 mM sodium lactate buffer pH 4.5. 2.5 mlaliquots of the standardized suspension of yeast are washed once bycentrifugation with sodium lactate buffer at 4,000 RPM and held as apellet pending the assay procedure as described below. A solution oftest substance and protein blank at the required concentration isprepared in 50/100 mM sodium lactate buffer pH 4.5 or sodium citratebuffer pH 4.5; unless otherwise stated, citrate or lactate buffers ontheir own were used as controls to determine full adhesion. Bovine serumalbumin (BSA) is used as a protein ‘blank’, Sigma-Aldrich A-7030; StLouis Mo., USA. The material is crude, “initial fractionation byheat-shock” as non-heat shocked fractions may contain active serumimmunoglobulin which may contribute to the inhibition of adhesion.

Washed yeast pellets from above are re-suspended in 2.5 ml volumes oftest, blank or control solutions and held at 37° C. for 10 minutespre-treatment. Each 2.5 ml volume of test, blank or control (with yeastin suspension) is then used to re-suspend a pellet of washed BECs fromabove. The combined suspensions are then incubated with gentle agitationat 37° C. for 60 minutes. The use of an orbital incubator at 50 RPMprovides a suitable environment.

After incubation, 2.5 ml of 50 mM sodium lactate buffer at pH 4.5 isadded to each of the test solutions mixed and subjected to a 5 secondpulse on a laboratory vortex. The purpose of the final dilution andvortexing is to separate loosely adhering yeast and yeast cells that arelying adjacent to, but not attached to, BECs. Further dilution may benecessary to facilitate counting of adherent yeast.

The assay may be performed as a pre-treatment of BECs by reversing theorder of re-suspension described above. BECs are first re-suspended in2.5 ml volumes of control buffer, test or protein blank, held at 37° C.for 10 minutes and then used to re-suspend a pellet of washed yeast andthe procedure completed as described above.

Assay of Microbicidal/Microbistatic Effect:

The assay is a standard microbiological suspension test wherein knownconcentrations of late log phase bacteria, yeast or fungi are inoculatedinto a fixed volume or weight of a test substance, blank or control.After a set period of time a neutralizing solution is added to stop theantimicrobial effect and the residual population of viablemicroorganisms is enumerated by serial dilution and plate counting. Thecounting procedure is a standard and basic microbiological procedure forenumerating viable microorganisms and will be well known to thoseskilled in the art.

In its generic form the method requires inoculation of 1 gram or 1 ml oftest sample with 0.1 ml of 18 hour (late log phase) followed by vigorousagitation to mix. After the predetermined exposure time has elapsed, 9.0ml of neutralizing buffer is added and mixed. This has the effect ofstopping the microbicidal effect which allows reliable estimates to bemade of the percentage kill achieved by a particular test sample in theperiod between inoculation and neutralization. Typically exposureperiods will range from 30 seconds up to 30 minutes and may progress toseveral hours where that time period is required to measure the effect.In order to enumerate residual viable cells and from that to computepercentage kill, serial dilutions and plate counts are carried out onthe 10 ml sample plus neutralizing buffer.

In the assays herein, stocks of bacteria, yeast and fungi are routinelystored on beads in 50% glycerol at −80° C. When required forviability/microbicidal assay, small aliquots from these stocks arespread on an appropriate nutrient agar, grown and sub-cultured to ensurepurity. Where broth cultures are required, 250 ml Erlenmeyer flaskscontaining 100 ml of broth are inoculated with a transfer loop from pureagar cultures and incubated under constant agitation in a rotaryincubator at 37° C.

Bacteria are routinely cultured using brain heart infusion (BHI) brothand agar or tryptone soya agar or broth (TSB), both of which may beacquired commercially from Oxoid, UK. TSB has the followingconstituents: 1.5% W/W tryptone (pancreatic digest of casein), 0.5% W/Wpeptone (papaic digest of soybean meal), 0.5% W/W sodium chloride, andagar at 1.5% W/W when required as a solid medium. Yeast are grown onYEPD agar or broth (see adhesion assay method hereinabove).

The diluting and neutralizing buffers used in the present method arephosphate buffered saline (PBS), containing 137 mM sodium chloride, 2.7mM potassium chloride and 10 mM phosphate, to which is added 3%polysorbate Tween 80 (anionic surfactant), 0.3% lecithin, and 0.5%histidine as neutralising agents. These ‘neutralising’ agents are thoseprescribed under EU Guidelines for ISO certification of microbicidalefficacy and were validated as suitable for neutralizing free fattyacids and chlorhexidine at the concentrations used herein.

Some microbial species are extremely difficult to culture, requiringspecialized media and/or anaerobic conditions which are not easilyachieved in liquid culture. Clostridium difficile for example is ananaerobe and also aero-intolerant dying off quickly in the presence ofoxygen. Fungal pathogens such as the Trychophyton species grow in hyphalmode and cannot be enumerated using the standard serial dilutionprocedure. In order to evaluate the microbicidal effect of the inventiveproduct against these species, agar dilution techniques were used andplate culture evaluated to find the minimum inhibitory concentration,i.e. that concentration of product above which no growth was observed.

The technique requires preparation of 10× concentrations of the testformulation for dilution in 9 volumes of agar. A 4 ml aliquot of this10× concentrate was added to 16 ml of cooled sterile agar and dispensedto a Petri dish—this represented the full strength (100%) formulation inthe agar medium, which may then be expressed as the percentageconcentration of microbicidal fatty acid in agar. Further dilutions ofthe 10× concentrate with sterile distilled water and the use of 4 mlaliquots of these dilutions in 16 ml of cooled agar allowed thepreparation of a series of decreasing concentrations of the product inagar. The test organisms were inoculated onto these agars with a sterileloop and the minimum inhibitory concentration was determined as thelowest dilution of test item at which no growth was detectable.

Inhibition of Biofilm Formation:

Bio-film is an attachment and accretion of planktonic bacteria, yeastand/or fungi to solid surfaces. The attachment is usually facilitatedthrough exo-polysaccharides secreted by the microorganisms and studiessuggest that it happens more easily on hydrophobic surfaces, such asplastic, and less easily on hydrophilic surfaces such as steel. Theformation of bio-film is highly significant in medical science,particularly its formation on the surface of indwelling catheters, whereit can be the source of catheter-related blood stream infections. Manydifferent microbial species are capable of forming bio-film, inmedicine. However, those of greatest significance are Staphylococcusaureus, Staphylococcus epidermidis, Enterococcus faecalis, Escherichiacoli, Klebsiella pneumoniae, Pseudominas aeruginosa and the yeast,Candida albicans.

The model of biofilm formation used herein is based on the method ofChristensen et al. J. Clin. Microbiol. 22: 996-1006, where growth of aselected organism and its biofilm formation in microtitre plate wells ismeasured by staining with crystal violet and measuring the intensity(Optical Density) of the stained biofilm in a microtitre plate reader at570 nm wavelength. The intensity of the stain is a measure of the extentof biofilm formation and its reduction in the presence of inhibitorysubstances is evaluated on the basis of reduction of the stainintensity.

The organism used in this assay is Staphylococcus aureus RN4220, arestriction deficient variant derived originally from NCTC 8325 knownfor its avidity in biofilm formation, particularly in the presence ofexcess levels of sodium chloride which is added to the culture media topromote that feature, culture media is 3.7% BHI from Oxoid UK to which4% W/V NaCl is added.

Nunc microtitre plate wells (Nunc International, Rosskilde, Denmark) arecoated with the test sample and 1:100 dilutions of late log phasebacteria in fresh saline supplemented BHI are dispensed to each testwell, and incubated for 24 hours to facilitate biofilm formation.

On completion of the incubation period, the wells are rinsed three timeswith sterile distilled water, and dried for one hour at 60° C. to fixthe adhering biofilm. Staining is achieved using a 0.4% solution ofcrystal violet which is added to each well and agitated there by rockingthe plate for 4 minutes. Excess dye is removed and the plates are rinsedthree times with sterile distilled water and dried. When dry, theintensity of the stain in each well is measured using a ASYS HitechUVM-340 plate reader at 570 nm (ASYS, Eugendorf, Germany).

Manipulation of Hemostasis: Clotting/Anti-Coagulation of Blood

Hemostasis is the inherent ability of the blood to react to traumaticdamage by forming a clot to plug the wound and so prevent excessive lossof blood and facilitate tissue repair and healing of the wound.Hemostasis also refers to the inherent ability of the blood to remainliquid in undamaged blood vessels and so fulfill its primaryphysiological transport functions. Both of these hemostatic aspects canbecome defective in disease and both can be overwhelmed by traumaticdamage, either accidentally or by default during surgical intervention.

The ability to manipulate the hemostatic mechanism by either amplifyingthe rate of clotting to prevent blood loss after accidental trauma or toprevent clotting during surgical repair and/or to prevent clot formationon, or in, surgically inserted medical instruments including catheters,is of great importance in medicine. To measure the rate of bloodclotting herein, use is made of a whole blood clotting meter, theHemochron Signature 11, manufactured by ITC, International TechnidyneCorporation, Edison, N.J., USA. The meter uses customized cuvettes intowhich one drop of fresh blood may be added and the time taken for clotformation is measured optically and reported electronically by thedevice.

In the assay herein, fresh sheep blood was used, which was drawn from ajugular vein using an 18 gauge needle and suitable volume syringe. Freshblood samples are immediately mixed with a volume of test material atvarying concentrations and one drop inserted in the cuvette. Sterilephosphate buffered saline is used as a blank to obviate the effects ofdilution and heparin is used as a control anti-coagulant. Measurement ofthe anti-clotting effects of the products of the invention are evidentin seconds compared to untreated whole blood, and compared to theanti-clotting effect of heparin and other proprietary anti-clotting(anti-coagulation) products. In optimal configuration the products ofthe invention achieve an anti-coagulation effect lasting in excess of1,000 seconds, which is comparable to 5,000 I.U of heparin under similartest conditions.

Measurement of accelerated clotting time (amplified clotting) is notpossible using the Hemochron device, as in most cases the products ofthe invention achieve clotting in under 60 seconds, which is ‘off scale’for the meter.

Comparative measurement of accelerated clotting times is achieved usingappropriate volumes of fresh blood mixed with a volume of test material,where the combined volumes totalled 5 ml in each case in a 15 mlgraduated Greiner centrifuge tube (Greiner Bio-one, North Carolina,USA). Immediately after addition of fresh blood, the tubes are invertedtwice to mix and allowed to stand without further agitation. Clotformation is visibly apparent when the tubes are tilted slightly, andonce a clot formed its integrity was such that it would remain suspendedwhen the tubes were inverted. In each test a 5 ml sample of fresh wholeblood (undiluted) is used as a control.

Analysis of Membrane Lipid and Free Fatty Acid

Routine analytical High Performance Liquid Chromatography is a suitablemethod of analyzing the individual membrane lipid components in acomposition such as lecithin. The procedure is well known to thoseskilled in the art of analytical chromatography. A Waters 2420 ELSD HPLCsystem from Waters Corporation, Milford, Mass., USA is used herein. ASymmetry C8 column (3.0×150 mm, 5 micron column is suitable with anon-linear gradient of 82% methanol in water containing 0.1%tri-fluoroacetic acid and gives adequate separation of membrane lipidcomponents.

Mammalian Cell Viability.

The reduction in viability of mammalian cells in the presence ofmembrane lipid emulsified free fatty acids was evaluated using Raji Blymphocytes grown in RPMI 1640 media containing 10% fetal calf serum andGibco Penstrep 15140 antibiotic supplement. Mature cells are harvestedby centrifugation at 1,000 RPM for 3 minutes and re-suspended in RPMI1640 without supplements for test purposes. Toxicity is assessed byevaluating uptake of trypan blue dye by dead cells using an InvitrogenCountess Automated Cell Counter (Invitrogen Inc, Carlsbad, Calif., USA).The procedure involves exposing a population of Raji B cells to the testsolution by mixing 100 μl of test and cell suspension in a microtitreplate well. After a pre-determined period of exposure, 10 μl of cell andtest mix are combined with 10 μl of 0.4% trypan blue and 10 μl of thisadded to the chamber of a cell cytometer cuvette and evaluated in thecell counter described above. Results are provided as a total cell countand numbers of these that are alive or dead are based on uptake orexclusion of the dye; a percentage viability is computed automatically.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the antimicrobial inhibitory effect of blood serumand the counteraction of this effect in combination with salts oforganic acids, as described in Example 3.

FIG. 2 illustrates blood clotting and anti-clotting properties ofvarying components of a product according to the invention, as describedin Example 5.

FIG. 3 illustrates anti-clotting properties of a catheter lockingsolution according to the invention, as described in Example 7.

FIG. 4 illustrates comparative microbicidal potency of a catheterlocking solution prepared according to the invention and compared withalternative commercial products, as described in Example 7.

FIG. 5 illustrates the biofilm inhibitory properties of a productaccording to the invention, as described in Example 8.

FIG. 6 illustrates the variable release of microbicidal effect achievedusing different membrane lipids and caprylic acid products according tothe invention, as described in Example 9.

FIG. 7 illustrates reduction in viability of Candida albicans inresponse to tailored release characteristics of products according tothe invention, as described in Example 10.

FIG. 8 illustrates inhibition of adhesion of Candida albicans bytailored release products according to the invention, as described inExample 10.

FIG. 9 illustrates the eradication of Staphylococcus aureus andStreptococcus pyogenes from an ex-vivo wound model, as described inExample 12.

FIG. 10 is a comparison of the ex-vivo efficacy of a product of theinvention in sodium citrate with conventional wound care antimicrobials,chlorhexidine and silver sulfadiazine, as described in Example 12.

FIG. 11 illustrates comparative eradication of established biofilm andthe superior efficacy of a product of the invention over existingcommercially available catheter locking solutions, as described inExample 13.

The practical applications and benefits of this invention areillustrated further in the following Examples. Unless otherwise stated,the membrane lipids used in the Examples are delipidised and containless than 3% conjugated extraneous lipid material as shown by HPLCanalysis as described in the Methods.

Example 1: Preparation of Adhesion Inhibitory Compositions of MembraneLipids

The following membrane lipids were purchased from Sigma Aldrich, Poole,Dorset, UK.

TABLE 3 Membrane lipids Compound/ Membrane lipid Component Originalsource Code Phospholipid Crude Lecithin Soya Bean LC PhospholipidPhosphatidylcholine Soya Bean PTC Phospholipid PhosphatidylethanolamineSoya Bean PTE Phospholipid Phosphatidylglycerol Soya Bean PTGPhospholipid phosphatidylinositol Soya Bean PTI PhospholipidPhosphatidylserine Soya Bean PTS Sphingolipid Ceramide Bovine Brain CSphingolipid Sphingomyelin Bovine Brain SGM GlycoglycerolipidMono-galactosyl Wheat flour MGDG diglyceride Cholesterol LanosterolSheep wool L

Crude lecithin was purchased from GR Lane Ltd, Gloucester, UK.

Aqueous dispersions of each of the above membrane lipids were preparedby suspending 1.0 grams in 100 ml of sterile distilled water (10 mg/ml)and agitating therein for 60 minutes with the aid of a magnetic stirrer.Dispersion of ceramide, sphingomyelin and lanosterol was achieved withthe aid of sonication and stirring, while dispersion of all othermembrane lipids was relatively easily achieved with stirring alone.Phospholipid and glycoglycerolipid dispersions were relatively stable,but others had a tendency to separate on standing and so all dispersionswere subjected to 30 seconds homogenization using a laboratoryhomogenizer immediately prior to use in the adhesion inhibitory assay. Asuitable homogeniser is an Ultra-Turax Model T18 (IKA Works, Wilmington,N.C. 28405, USA) fitted with an S18N-19G dispersing tool, operating at6,000 to 8,000 RPM.

The adhesion inhibitory properties of each phospholipid were tested asdescribed in the methods at concentrations of 10, 5, and 2.5 mg/ml; thelower dilutions were achieved by diluting the original dispersion withsterile distilled water. Due to logistical limitations of collectingsufficient buccal epithelial cells at any one time, the assays wereconducted in blocks of five individual membrane lipids on separate days,and the results shown in Table 4 are a composite of these; the Controlis zero concentration of test item and Bovine Serum Albumin (BSA) isused as a protein blank.

TABLE 4 Candida albicans per 100 Buccal Epithelial Cell % inhibitionItem Code 0 mg/ml 2.5 mg/ml 5.0 mg/ml 10 mg/ml at 2.5 mg/ml LC 540 387291 92 28 PTC 540 127 59 0 76 PTE 496 167 74 0 66 PTG 496 114 62 0 77PTI 519 201 87 0 61 PTS 519 233 102 39 55 C 484 363 282 109 25 SGM 484379 277 126 21 MGDG 540 261 128 66 51 L 519 306 236 133 41 BSA 496 400379 303 19

In preliminary evaluation of crude lecithin from various sources (eggyolk, soya and sunflower), it was noted that the adhesion inhibitoryproperties varied considerably, despite the reported purity beingapproximately the same. It was reasoned that the purchased materialmight contain extraneous fat or tri-glyceride and to remove this, theoriginal material was suspended in acetone at 10% W/V and stirred for 30minutes after which the insoluble membrane lipid was allowed to settleand the acetone decanted. The ‘de-lipidised’ lecithin (DLL) was dried ina fume hood, and its adhesion inhibitory properties evaluated aspreviously described. The remaining membrane lipids listed in Table 4were delipidised in the same manner. The delipidised membrane lipidswere analysed by HPLC as described in the Methods, and all were shown tocontain less than 3% conjugated extraneous lipid material. The resultsare shown in Table 5 below, including the incremental increase inadhesion inhibitory effect achieved by de-lipidisation of individualmembrane lipids.

TABLE 5 Candida albicans per 100 Buccal Epithelial Cell % inhibitionItem Code 0 mg/ml 2.5 mg/ml 5.0 mg/ml 10 mg/ml at 2.5 mg/ml LC 540 387291 92 28 DLL 489 138 48 0 71 (+43) DL PTC 508 132 0 0 84 (+8)  DL PTE515 139 0 0 73 (+7)  DL PTG 474 57 0 0 88 (+11) DL PTI 519 68 0 0 77(+16) DL PTS 519 145 62 0 72 (17)  DL C 493 305 118 41 38 (+13) DL SGM511 301 133 44 41 (+20) DL MGDG 516 206 116 0 60 (+9)  DL L 536 247 9149 54 (+13) DL BSA 513 431 393 329 16 (−3) 

Solvent de-lipidisation increases the adhesion inhibitory properties ofmembrane lipids, especially lecithin by a factor of 2.5, bringing it inline with that of its individual components: PTC, PTE, PTG, PTI and PTS.Further de-lipidisation of the individual components of lecithinachieves further improvement in the adhesion inhibitory properties ofthese, although less dramatic compared to the effect achieved inde-lipidising crude lecithin. It is assumed here that the commercialprocess of isolation used to extract individual components of lecithinresults in almost complete de-lipidisation, hence the optimal inhibitoryeffect of these relative to the ‘crude’ lecithin and the smallerincremental effect achieved by additional de-lipidisation.

In many of the applications for the product of this invention theadhesion inhibitory substance will be in contact with blood serum, mucusand other body fluids. It has been discovered that de-lipidised lecithinand most of its constituents lose their adhesion inhibitory propertiesin the presence of Bovine Serum Albumin (BSA). It has also beendiscovered however, that adding an organic acid salt counteracts thisnegative effect and restores most of the adhesion inhibitory propertiesto the combined membrane lipid and BSA.

In this example, 100 mM solutions of sodium lactate and sodium citratewere used. The salts were prepared first as 200 mM solutions and the pHadjusted to 4.5, and aliquots of both were used to prepare 1% solutionsof bovine serum albumin (i.e. 1% BSA in 200 mM sodium salt of citrate orlactate at pH 4.5). The salt solutions were used to dilute a 1%suspension of de-lipidised lecithin (DLL) and a 1% suspension ofphosphatidyl choline (PTC), to achieve preparations of 0.5% DLL and 0.5%PTC in 100 mM sodium lactate/sodium citrate at pH 4.5 with 5% BSA ineach. A further dilution of the membrane lipid suspension to 0.5% withwater and the use of this in halving dilutions of salt solutioncontaining 0.5% BSA gave a composition of 0.25% membrane lipid in 100 mMsalt with 0.25% BSA. Similar procedures were used to prepare 0.25% and0.5% solutions of BSA in water and in 100 mM organic salt solution: theresults are presented in Table 6.

TABLE 6 Candida albicans per 100 Buccal Epithelial Cell % inhibitionItem Code 0 mg/ml 2.5 mg/ml 5.0 mg/ml 10 mg/ml at 5 mg/ml LC: water 540387 291 92 46 DLL: water 489 138 48 0 90 DLL + BSA 396 320 280 ND 30water DLL + BSA 396 290 144 ND 63 Sod' Citrate DLL + BSA 396 340 164 ND58 Sod' Lactate PTC: water 540 127 59 0 90 PTC + BSA 474 392 344 ND 28water PTC + BSA 474 273 162 ND 66 Sod' citrate PTC + BSA 474 291 138 ND70 Sod' Lactate BSA 474 393 369 ND 22 Sod' citrate BSA 474 408 372 ND 21Sod' Lactate BSA: water 496 438 400 362 12

In this Example, with the exception of crude lecithin, all of themembrane phospholipids tested were shown to have superior inhibitoryproperties preventing Candida albicans adhesion to Buccal EpithelialCells. When de-lipidised with acetone, crude lecithin is as inhibitoryas its main membrane phospholipid components. In combination with bovineserum albumin, the adhesion inhibitory property is eradicated, but itcan be restored in the presence of 100 mM solutions of organic acidsalts at acid pH.

Example 2

Preparation and Testing of Microbicidal Combinations of Membrane Lipidand Free Fatty Acid

The microbicidal effect of 0.5% caprylic acid in 0.4% de-lipidisedlecithin, prepared as described in the Methods, against a range ofmicrobial species may be demonstrated using the microbicidal suspensiontest described in the Methods. The potency of this material is such thatcomplete eradication of an inoculum in excess of 6 logs may beanticipated in less than 6 minutes. Gram negative species are slightlymore resistant than gram positives, particularly those known to be slimeproducers, such as Escherichia coli and Pseudomonas fluorescens, whereit is thought that the slime layer protects from contact with the fattyacid for some additional minutes.

Table 7 below illustrates the reduction in viability of a range ofbacteria and yeast. Because the inoculums vary, the latest time toachieve 50% in viability of the inoculum is provided in the last column(L. T. 50%), as a comparative measure of potency against that particularspecies.

TABLE 7 Microbicidal Effect: 0.5% Caprylic in 0.4% DLL Exposure TimeL.T. Organism Gram 0 60 120 180 240 300 360 50% Staph aureus RN4220 +ve8.53 5.22 3.71 1.61 0 0 0 <120 Staph epidermidis +ve 8.27 4.96 2.67 0 00 0 <120 NCTC 11047 Strep pyogenes +ve 7.91 5.32 3.33 1.49 0 0 0 <120NCTC 8198 Strep faecalis +ve 8.11 4.98 2.54 1.66 0 0 0 <120 NCTC 12697E. coli −ve 8.43 7.17 6.2 5.28 3.64 1.79 0 <240 ATCC 11698 Salmonella−ve 7.76 5.94 3.52 2.27 0 0 0 <120 typhimurium NCTC 74 Klebsiellaaerogenes −ve 6.93 5.15 3.37 1.99 0 0 0 <120 NCTC 9528 Proteus mirabilisC.I. −ve 7.41 6.61 5.89 3.75 1.59 0 0 <240 Enterobacter Cloacae −ve 8.626.54 4.94 2.32 0 0 0 <180 C.I. Pseudomanas −ve 8.33 7.13 5.48 4.78 2.921.23 0 <240 aeruginosa ATCC 27853 Pseudomanas −ve 7.29 6.42 5.13 3.631.29 0 0 <240 fluorescens NCTC 10038 Candida albicans C.I. Yeast 6.864.39 2.96 0 0 0 0 <120 Candida glabrata Yeast 6.74 4.19 2.23 0 0 0 0<120 NCPF 8750 Crypto' neoformans Yeast 5.97 4.11 2.96 0 0 0 <120 C.I.Note C.I = Clinical Isolate Note: L.T 50% is Latest Time to achieve 50%reduction in viability in this test

The numerical data presented are log numbers where the integer is thelog and the decimal the colony count at that log: 6.3×10⁵ for example ispresented as 5.63 and this convention will be used throughout thisdocument.

For reasons of difficulty in culturing or because of special growthrequirements the potency of the formulation against anaerobes isdetermined by the Minimum Inhibitory Concentration procedure asdescribed in the Methods, and the results are presented in Table 8.

TABLE 8 Minimum Inhibitory Concentration of 0.5% Caprylic in 0.4% DLLagainst anaerobes and fungal pathogens using agar dilution technique:MIC value is the % of Caprylic acid in the test plate Organism Notes MICClostridium perfringens G −ve anaerobe >0.1 ATCC 43150 Clostridiumdifficile ATCC G −ve anaerobe (aero intolerant) >0.1 43598 Bacteroidesfragilis ATCC G −ve obligate anaerobe >0.2 43859 Fusobacterium nucleatumG −ve anaerobe: >0.3 NCTC 10652 3 weeks Columbia blood agarDesulfovibrio G −ve anaerobe >0.1 desulfuricans Porphyromonas gingivalisG −ve obligate anaerobe >0.1 Campylobacter jejunii G −vemicroaerophilic >0.075 Actinobacillus G −ve microaerophilic >0.1actinomycetemcomitans Corynebacterium G +ve facultative anaerobe >0.1diphtheriae Treponema denticola Obligate anaerobe >0.2 MycobacteriumAerobic acid fast bacillus 6 weeks >0.2 tuberculosis onLowenstein-jensen medium

Example 3: Membrane Lipid Antimicrobials in Blood Contact Applications

In common with the negative effect on adhesion inhibitory propertiesreported in Example 1, the presence of bovine serum albumin also impactson the microbicidal effect. However as in Example 1, this can beovercome by incorporation of an organic acid salt, at acid pH.

An oil-in-water emulsion of 0.5% caprylic acid in 0.4% de-lipidisedlecithin is used in this Example. 200 mM solutions of glycolic, acetic,lactic, and citric acids were adjusted to pH 4.5 using 200 mM sodiumhydroxide. 10% W/V solutions of Bovine Serum Albumin were prepared ineach of the organic acid salts and in water as a control. 5 ml aliquotsof the 0.5% caprylic emulsion were added to 5 ml aliquots of each saltsolution with and without BSA, and in water with and without BSA,resulting in dispersions of 0.25% caprylic emulsion in 0.2% de-lipidisedlecithin dispersed in 100 mM salt solution at pH 4.5 in each testsample.

E. coli was selected for this Example as it has been demonstrated to beone of the most resistant species. The bacterium was cultured in BrainHeart Infusion broth for 18 hours at 37° C. 10 ml fractions of each testsample were inoculated at time zero with 1.1 ml of the overnightculture, and 1.0 ml samples removed from this at 3 minute intervals forresidual viability determination using serial dilution and platecounting procedures.

TABLE 9 Antimicrobial effect is impaired by blood components andre-instated by combination with salts of organic acids 0.25% Caprylic in0.2% De-lipidised Lecithin +/− 5% Bovine Seum Albumin kill 0 3 6 9 12 1518 21 24 27 30 time Water 8.37 4.97 2.23 0 0 0 0 0 0 0 0 9 Water + BSA8.37 7.51 6.42 5.98 5.17 4.93 3.72 3.33 2.21 1.49 0 30 Glycolate 8.375.25 2.6 8 0 0 0 0 0 0 0 0 9 Glycolate + 8.37 6.62 5.35 3.79 2.41 1.56 00 0 0 0 18 BSA Acetate 8.37 5.94 4.21 1.28 0 0 0 0 0 0 0 12 Acetate +8.37 7.41 6.93 5.24 4.53 3.47 2.18 1.34 0 0 0 24 BSA Citrate 8.37 4.92.7 0 0 0 0 0 0 0 0 12 Citrate + BSA 8.37 6.2 4.5 1.93 0 0 0 0 0 0 0 12Lactate 8.37 5.29 3.19 0 0 0 0 0 0 0 0 9 Lactate + 8.37 7.1 4.9 1.8 0 00 0 0 0 0 12 BSA

In a water diluent the effect of 5% W/V BSA is to reduce potency of0.25% caprylic acid by a factor of 3: Kill Time is extended from 9minutes to 27. In combination with 100 mM sodium salts of citrate andlactate acids at pH 4.5, the kill time is 12 minutes, glycolate is 18minutes and acetate is 24. The results of combination with citrate andlactate acid salts with and without BSA are presented in Table 9 aboveand illustrated in FIG. 1.

Example 4: Use in Food Safety

In this Example 1 cm³ sections of fresh beef were deliberatelycontaminated with late log phase cultures of Salmonella enterica andEscherichia coli O157:H7 at room temperature and allowed to adhere therefor 60 minutes. Confirmation of contamination was obtained bymechanically macerating the meat sections in sterile phosphate bufferedsaline (PBS) and enumeration by serial dilution and plate counting.

A suitable but not optimal carcass wash was prepared using 0.5% caprylicacid in 0.4% de-lipidised lecithin and diluting that ×2 with 200 mMsodium citrate at pH 4.5 as described in Example 3. The wash was sprayedonto contaminated sections of fresh beef, and the antimicrobial effectevaluated by mechanical maceration, serial dilution and plate counting.The results are presented in Table 10 below:

TABLE 10 Reduction in Viability of Pathogens on Fresh Beef TimeEscherichia coli Salmonella enterica minutes Untreated Treated UntreatedTreated 0 7.14 6.96 6.91 6.61 30 6.98 5.61 6.37 4.59 60 6.56 3.89 6.717.73 90 6.82 2.19 6.52 1.29 120 6.69 ND 6.28 ND Note: ND = Not detected

It should be appreciated that the rate of kill on a porous surface suchas meat is extended due to the nature of the surface and the timerequired to permeate it.

Example 5: Use of Membrane Lipids to Manipulate Blood Clotting Time

As described in this Example the membrane lipid products used may betailored to affect the rate of blood clotting in addition tocontributing a significant antimicrobial effect.

Rate of blood clotting was determined using freshly aspirated sheepblood and apparatus and procedures described in the Methods section. Anactivated blood clotting meter was used to measure anti-clotting effectsand a visual tube comparison to evaluate reduced clotting times.

It was unexpectedly discovered that an aqueous dispersion ofde-lipidised lecithin (DLL) will amplify the rate of blood clotting in aconcentration dependent manner. Varying concentrations of dispersions ofDLL were prepared by suspending the required weight in a volume ofwater, stirring for 30 minutes to hydrate and homogenizing thesuspension with a laboratory homogenizer.

Blood clotting and/or anti-clotting effects were assessed using a 20%ratio of test item to fresh blood: in practice 4.0 ml of fresh sheepblood was added to tubes containing 1.0 ml of test item, inverted twiceto mix and then either left standing for visual assessment of reducedclotting time or a single drop was applied to the cuvette of anactivated blood clotting apparatus for assessment of anti-clottingeffect (extended time to clot formation).

As the concentration of DLL in the test item increases, the observedrate of blood clotting increases, i.e. time to clot formation decreasesfrom 360 seconds for whole blood to approximately 60 seconds or less atDLL concentrations in excess of 1%.

The addition of caprylic acid by emulsification in DLL will also amplifythe clotting effect up to a point where the concentration by weightequals or slightly exceeds the concentration of DLL by weight. At 0.5%DLL on its own, the clotting time is approximately 125 seconds. Additionof emulsified caprylic acid up to 0.5% does not significantly affect theclotting time. Between 0.5% and 0.75% caprylic acid there is a furtherdepression of clotting time from 125 seconds to approximately 40 seconds(70% less). Thereafter, however, the anti-clotting effect is reversedand clotting time increases with increasing concentration of caprylicacid.

When the concentration of DLL is reduced to 0.25% with increasingconcentration of caprylic acid, there is no significant reduction ofclotting over and above that attributable to DLL on its own. In fact, asthe concentration of caprylic increases to between 0.75% and 1.0%, theclotting time is restored to normal (360 seconds). Further increasingthe relative ratio of caprylic acid in 0.25% DLL, has an anti-clottingeffect.

Using an emulsion of 0.25% caprylic acid in 0.25% DLL as a standard(STD), it can also be observed that addition of increasingconcentrations of sodium citrate salts at pH 4.5 suppresses theanti-clotting effect of DLL in combination with caprylic acid. At 0.5%concentration, the anti-clotting effect of 0.25% caprylic acid in 0.25%DLL has been suppressed completely and clotting time has been restoredto above normal (400 seconds). A further increase of sodium citrate inthis composition has a progressively increasing anti-clotting effect—upto 600 seconds at 2% sodium citrate in 0.25% caprylic emulsified in0.25% DLL. The results are illustrated in FIG. 2.

Example 6: Use of Antimicrobial Membrane Lipids in Amplified Clottingfor Wound Care

The combined antimicrobial and clot forming capability of the productsof the invention are demonstrated in this Example. A suitable, exampleof an antimicrobial membrane lipid preparation with enhanced clotforming properties may be selected from the combinations prepared inExample 3. A 0.5% dispersion of DLL in sterile distilled water with 1.0%caprylic acid was used here. It will be noted that no organic acid saltis included in this example and further noted that the absence of suchreduces the antimicrobial potency but does not limit the clottingeffect. The use of a relatively high caprylic acid load compensates forthe interference of blood components with the antimicrobial effect.

A bacterial inoculum of E. coli was grown in Brain Heart Infusion brothfor 18 hours and a 10 ml volume of this was sedimented by centrifugationat 4,000 RPM for 10 minutes, the pellet was re-suspended in 1.0 ml ofthe supernatant (concentration ×10).

Two 4.0 ml samples of fresh blood were dispensed to two 15 ml Greinercentrifuge tubes and both were inoculated with 0.1 ml of concentrated E.coli suspension: a blank comprising 5.0 ml of 5% Bovine Serum Albumin insterile distilled water was inoculated at the same time.

A 1.0 ml of volume of 400 I. U. heparin and 500 I. U. streptokinase inwater was added to the first tube, and 1.0 ml of test preparation to thesecond. Both tubes were mixed by inversion and incubated at 37° C. for45 minutes. The tube containing the test preparation clotted inapproximately 60 seconds; no clot was detectable in theheparin/streptokinase tube after 45 minutes.

At the end of the 45 minute incubation a 1.0 ml of volume of 400 I. U.heparin and 500 I. U. streptokinase in water was added to the clottedtube with test preparation and 1.0 ml of sterile distilled water addedto the second. Both samples were homogenized at 1,000 RPM for 2 minutesusing an Ultra Turax homogenizer. The clot disruption procedure tookapproximately 15 minutes (60 minutes exposure in total), after whichserial dilution and plate counting procedures were used to enumerateresidual bacterial viability in all three tubes.

The BSA blank contained 6.4×10⁷ viable E. coli cells per ml, the controlblood (heparin/streptokinase treated) sample contained 8.3×10⁵, and noviable bacteria were recovered from the test sample.

It will be clear to those skilled in the art that preparations such asdescribed in this Example may be applied to wounds in the form of aliquid, spray, gel, powder, or wet-bandage. It will also be clear tothose skilled in the art that the preparations described may be added toother pro-coagulants such as chitin, kaolin or alginate to enhance theirpro-coagulation effect and add a microbicidal effect.

Example 7: Use of Antimicrobial Membrane Lipids with Anti-ClottingEffect in Surgical Procedures

Ingress of potentially infectious agents during surgical procedures is amajor cause for concern among healthcare professionals. The use ofirrigating fluids with antimicrobial effects facilitates prevention ofthis. There are also many surgical procedures where the ability toprevent blood clotting is advantageous, micro-surgery procedures forexample, where pre-emptive clotting may be exacerbated by the implementsused and where the use of an irrigating solution to wash out blood andbody fluids to prevent occlusion of the site is desirable. A specializedapplication is the use of anti-clotting liquids to fill the void volumeof indwelling catheters during periods when the catheter is not in use.

In this Example, a dispersion of 0.4% de-lipidised lecithin (DLL) with0.5% caprylic acid emulsified therein is prepared as described in theMethods. The emulsion is diluted to 50% of its initial concentrationwith 200 mM sodium citrate at pH 4.5, the result being 0.25% caprylicacid, 0.2% DLL in 100 mM sodium citrate, or approximately 2.5% W/Vsodium salt of citric acid at pH 4.5. Sodium citrate is prepared byadjusting the pH of 200 mM citric acid with 200 mM sodium hydroxide to4.5; this is not the same as ‘tri-sodium citrate’ which is commonly usedas an anti-clotting agent, because not all of the carboxylic acidresidues have been salted out.

As described hereinabove, the use of a viscosity-enhancing agent toadjust the viscosity of the formulation to approximate to that of wholeblood provides a distinct advantage in preventing dilution of a catheterlocking solution at the catheter tip due to blood flow turbulence.Dextran 40 is used here as a viscosity-enhancing agent where it has beenfound that 20% W/V inclusion provides a viscosity of approximately 4 cP,the viscosity of human blood being between 3.6 and 6 cP.

The emulsified free fatty acid/membrane lipid catheter locking solutionused here (ML CLS) has the following constituents: 0.2% W/V de-lipidisedlecithin; 0.25% caprylic acid; 20% dextran 40, in 100 mM (2.5% W/V)sodium citrate, pH 4.5. Aliquots of this formulation were dispensed to15 ml Greiner centrifuge tubes in the following amounts: 0, 0.25, 0.5,0.75 and 1.0 ml amounts. To each of these 5.0, 4.75, 4.5 and 4.25 mlaliquots of fresh sheep blood were added, respectively. Each tube wasevaluated consecutively with fresh blood added immediately after it wasaspirated. The respective volume dilutions represent 0, 5%, 10%, 15% and20% by volume of blood. Immediately after addition of blood, the tubeswere inverted twice to mix and a single drop added to the test well of aHemochron Signature Activated Blood Clotting Meter.

A similar procedure was adopted using a solution of 25,000 I.U. ofHeparin, which at 5%, 10, 15% and 20% gave sample concentrations of1,250, 2,500, 3,750 and 5,000 units per ml of test volume.

Also tested were a commercially available Catheter Locking solution,Duralock from MedComp, which contains 47% sodium citrate only, and acomposition of 0.05% Methylene Blue, 0.15% Methyl Parabens, and 0.015%Propyl Parabens in 7% (0.24M) sodium citrate, being a replica of thereported formulation for Zuragen; and Taurolock from Tauropharm AGcomprising 1.35% Taurolidine in 4% sodium citrate: (see Table 2).Phosphate Buffered Saline (PBS) is used as a dilution control.

It should be noted here that the Hemochron blood clotting system relieson a clot activation process which accelerates time to clot formation;whole blood without additives clots in less than 200 seconds in thisapparatus. The timelines in this experiment are not directly comparabletherefore with clotting times reported in Example 5 where the baselinefor normal clot time is shown as 360 seconds being the observed time fornormal (non-activated) clotting. It should also be noted here that theHemochron meter goes ‘out of range’ at 1,000 seconds of ActivatedClotting Time and more extended time recordings are not available.

The results are reported in table 11 below and illustrated in FIG. 3.

TABLE 11 Activated Blood Clotting Times for Various Catheter LockingSolutions % Incorporation in whole blood 0% 5% 10% 15% 20% ML CLS 159347 537 769 1018 Heparin ¹ 159 284 423 601 1023 Duralock 159 300 364 484722 Zuragen 159 219 265 394 614 Taurolock 159 230 310 380 510 PBScontrol 159 163 165 233 296 Note ¹ Heparin concentrations range from1,250 I.U. at 5% to 5,000 I.U. per ml at 20%

In terms of metered anti-clotting effect, the inventive product of thisExample is better than 25,000 I.U. of Heparin and considerably betterthan Duralock, Zuragen or Taurolock. It should be noted again however,that these are ‘Activated’ clotting times. In practice, none of thetreated samples—apart from control PBS—showed any visual sign ofclotting even after several hours.

The antimicrobial effect of the formulation in this Example was testedusing procedures similar to those used in Example 6, with the exceptionthat the clot disrupting agents, heparin and streptokinase, were used tobreak the clots in the control untreated bloods.

18 hour Brain Heart Infusion Broth cultures of Staphylococcus aureus,Streptococcus epidermidis, Escherichia coli and an 18 hour culture ofCandida albicans grown in Yeast Extract Peptone Dextrose broth wereconcentrated ×10 by centrifugation and re-suspension in one tenth volumeof supernatant.

2.5 ml aliquots of the bacterial concentrates were used to inoculate22.5 ml volumes of freshly aspirated sheep blood, mixed and the blooddispensed as 5.0 ml, 4.75 ml, 4.5 ml, 4.25 ml and 4.0 ml volumes toGreiner centrifuge tubes containing 0, 0.25 ml, 0.5 ml, 0.75 ml and 1.0ml volumes of the test formulation of this Example.

The inoculated tubes were incubated for 45 minutes at 37° C. followingwhich time, a 1.0 ml of volume of 400 I. U. heparin and 500 I. U.Streptokinase in water was added to all tubes and each was subjected toslow speed homogenization at 1,000 RPM for 2 minutes: the only visibleclotting was in the control tubes. Immediately after 60 minutes hadelapsed, serial dilution and plate count methods were used to assessresidual viability in all samples. For comparative purposes, the sameprocedure was repeated with Taurolock, Duralock, Zuragen and Heparin atthe maximum dose loading of 1.0 ml in 4.0 ml of blood only. The resultsare presented in Table 12 and illustrated in FIG. 4.

TABLE 12 Comparative Microbicidal Effect of Catheter Locking Solutionsin whole blood Escherichia Staphylococcus Streptococcus Candida coliaureus epidermidis albicans Time zero 7.69 8.71 8.12 6.67 Blank Time8.19 8.9 8.51 6.33 60 min ML CLS 4.83 3.62 4.17 3.9 5% ML CLS 2.58 1.441.97 0 10% ML CLS 1.62 0 0 0 15% ML CLS 0 0 0 0 20% Taurolock 6.53 4.154.72 5.72 20% Duralock 6.97 6.49 7.78 6.54 20% Zuragen 5.56 4.91 5.734.28 20% Heparin 7.92 8.39 8.22 6.94 25,000 I.U. 20%

In this test the 20% by volume ML CLS according to the inventionachieved complete eradication of E. coli (8 logs), Staph aureus (8logs), Strep epidermidis (8 logs) and Candida albicans (6 logs) in onehour in whole blood: a 5% volume achieved approximately 4 log reductionof the test inoculums in the same time. Of the four comparativepreparations (Duralock, Taurolock, Zuragen or Heparin), only Zuragen andTaurolock had an appreciable microbicidal effect achieving a reductionof between 3 and 4 logs in viability of the test inoculum in one hour;Duralock appears to have a microbistatic effect while no microbialinhibition could be attributed to Heparin.

Thus, the above catheter locking solution according to the inventionexhibits significantly better anti-clotting effects and much greatermicrobicidal effect than existing conventional products.

Example 8: Use of Membrane Lipids in Antimicrobial Surface Coating

A suitable method of applying a persistent coating of a productaccording to the invention involves emulsification of 1% caprylic acidin 0.8% de-lipidised lecithin prepared as described in the Methods. Anequivalent volume of 100 mM sodium citrate buffer pH 4.5 is added to theemulsion to obtain a final concentration of 0.5% caprylic acid, 0.4%de-lipidised lecithin in 50 mM sodium citrate. Eight volumes of absoluteethanol are then added slowly to two volumes of the emulsion withconstant vigorous stirring to make the final coating material in 80%ethanol.

In order to coat a plastic surface it is preferable to use some form ofsurface conditioning which may include a process known as coronadischarge wherein an electrical field is generated across the surfaceimparting a residual charge which facilitates adhesion of the appliedcoat. Following corona treatment, the ethanol solution described aboveis sprayed on the surface and dried under forced air conditions at 60°C. Several coats may be applied to construct a layer of antimicrobialcoating.

A base layer of membrane lipid may first be applied to an inert surface,and once dried and annealed it is used to ‘anchor’ a second layer ofde-lipidised membrane lipid emulsified free fatty acid according to theinvention.

In this Example an organic solvent solution of a membrane lipid isapplied to the surface of a microtitre plate well, dried and fixed byheating at 60° C. followed by a further application of an aqueoussuspension of caprylic acid emulsified in de-lipidised lecithin preparedas described in the Methods. The de-lipidised lecithin emulsion wasdried and annealed to the first lecithin coating by heating at 60° C.for 3 hours. Plates treated with de-lipidised lecithin, without caprylicacid were used as a control and untreated plates were used to determineoptimum biofilm formation.

A base layer of de-lipidised lecithin (DLL) was prepared by suspending5% by weight DLL in 80% ethanol:water, and dispensing 100 μl aliquots ofthis to the test wells. Wells for determination of optimum biofilm wereleft untreated. The ethanol fractions were dried in a fume hood andannealed at 60° C. for one hour in an oven. 1% W/V dispersion ofde-lipidised lecithin was prepared in sterile distilled water asdescribed in Example 1, and a volume of this used to emulsify 0.25%caprylic acid. 100 μl aliquots of DLL or DLL+0.25% caprylic weretransferred to DLL, pre-coated wells and dried in an oven at 60° C. for3 hours.

A 10 hour culture of Staphylococcus aureus RN 4220 grown in Brain HeartInfusion broth (BHI), supplemented with 4% sodium chloride was used asinoculum. The mid log phase culture was diluted to 1% with fresh sodiumchloride supplemented BHI and 200 μl of this added to the microtitreplate wells, covered and incubated.

At each sample point, 100 μl from each well was transferred to a freshplate for assessment of growth by Optical Density at 570 nM and theplate was then decanted and washed with copious volumes of steriledistilled water, dried and annealed in an oven at 60° C.

When dry, 100 μl of 0.4% crystal violet was added to each well—includinguntreated controls. After 4 minutes, excess crystal was drained off, andthe plates were again washed with copious volumes of water to removeexcess dye and again dried with the aid of an oven at 60° C. The resultsare presented in table 13 and illustrated in FIG. 5.

TABLE 13 Inhibition of Biofilm Formation: 24 hour culture OpticalDensity 570 nM Control growth 2.8 Coated planktonic growth 2.4 Uncoatedbiofilm 0.9 DLL coated biofilm 0.7 DLL + Cap Inhibited biofilm 0.1

Uncoated planktonic growth is largely unaffected by the coating ofinhibitory DLL+caprylic acid. Wells coated with DLL alone (DLL coatedbiofilm) are slightly reduced, but in comparison, wells coated with theproduct of this invention (DLL+Cap Inhibited Biofilm) are essentiallyfree of any biofilm: the coating itself takes up some of the crystalviolet dye which accounts for a small increase in Optical Density in theDLL+Cap wells.

Example 9: The Use of Membrane Lipids to Achieve Sustained Release ofMicrobicidal Free Fatty Acids

In therapeutic applications considerable advantage may be gained fromusing combinations of membrane lipid emulsions with ‘tailored’ releasecharacteristics, which facilitates sustained microbicidal effect at theepithelial surface.

In this Example individual membrane lipids were selected from each ofthe four classes presented in Table 1 and were the same as those used inadhesion inhibitory studies in Example 1. 0.4% Aqueous dispersions ofeach were prepared and 0.2% caprylic emulsified in each using proceduresdescribed in the Methods for de-lipidised lecithin (DLL). A 0.4% sampleof DLL with 0.2% caprylic was also prepared. Each of the membrane lipidpreparations was diluted to 50% of its volume with 200 mM sodium citrateat pH 4.5, and therefore each preparation then consisted of 0.1%caprylic acid and 0.2% membrane lipid in 100 mM sodium citrate at pH4.5. It should be noted here that this is less than half of themicrobicidal caprylic acid content of the test item used in Example 3,Table 9.

The yeast Candida albicans was used in this Example, and the inoculumprepared as an 18 hour YEPD broth culture as described in the Methods.The late log phase culture was centrifuged at 4,000 RPM for 5 minutesand re-suspended in one tenth volume of supernatant to concentrate ×10.

12.0 gram samples of each test preparation were dispensed to Sterilintubes and each of these was inoculated with 1.2 ml aliquots ofconcentrated yeast culture. Samples of 1.0 ml volume were withdrawn formthese inoculated tubes at timed intervals over the course of one hourand added to 9.0 ml of diluting buffer containing 3% Tween 80 toneutralize. Serial dilutions and plate counting was undertaken toenumerate residual viability as described in the Methods. The resultsare presented in Table 14 below and illustrated in FIG. 6.

TABLE 14 Variable Release Characteristics of Membrane Lipids: Kill timefor >6 logs; Candida albicans Minutes 0 5 10 15 20 25 30 35 40 45 50 5560 Blank 7.13 6.92 6.86 6.69 7.19 6.67 6.93 7.21 6.73 7.17 6.89 6.786.84 DLL 6.39 5.64 4.53 3.21 1.96 0 PTC 6.5 4.84 2.57 1.22 0 0 PTE 7.325.41 3.67 1.88 0 PTG 6.76 5.93 5.17 4.53 3.68 2.82 1.95 1.14 0 PTI 7.416.11 4.83 3.79 2.32 1.16 0 PTS 6.8 6.36 5.85 5.23 4.53 3.79 3.13 2.611.88 1.1 0 C 7.24 7.28 6.89 6.54 6.14 5.96 5.32 4.97 4.65 4.29 3.87 3.743.32 SGM 6.84 6.55 6.12 5.85 5.42 4.87 4.22 3.76 3.13 2.79 2.22 1.841.33 MGDG 7.3 6.66 6.1 5.16 4.34 3.32 1.84 0 L 7.39 6.45 5.86 5.34 4.784.21 3.67 2.98 2.33 1.74 1.17 0

It is evident from the results that the slowest acting emulsions areCeramide and Sphingomyelin followed by Lanosterol, Phosphatidylserineand Phosphatidylglycerol; the fastest acting emulsions arePhosphatidylcholine, Phosphatidylethanolamine and the combination ofphospholipids in de-lipidised lecithin (DLL).

Example 10: The Use of Combinations of Antimicrobial Membrane LipidEmulsions to Fortify Mucus and Achieve a ‘Tailored’ Microbicidal Effectat the Mucosal Surface

Mucosal fortification involves complementary hydration, lubrication andenhanced antimicrobial effect of mucosal secretions of the eye, nose,mouth, naso-pharyngeal surfaces, the gastro-intestinal tract and thegenitalia. This Example describes a preparation suitable forfortification of the mucosal secretions of the mouth and vagina and mostparticularly suitable for use by individuals susceptible to recurringoral and/or vaginal thrush and other common bacterial and viralinfections responsive to the products of this invention.

Examples of Mucosal Fortificants

-   Part A: A fast acting membrane lipid emulsion of caprylic acid (Cap)    is based on 0.2% W/V Phosphatidylcholine (PTC) dispersed in sterile    distilled water, hydrated and homogenized as described in the    Methods and used to emulsify 0.25% W/V caprylic acid by the    procedure described.-   Part B: A slow acting membrane lipid emulsion of caprylic acid (Cap)    is based on 0.2% Sphingomyelin (SGM), dispersed, hydrated and    homogenized as described and then used to emulsify 0.25% W/V    caprylic acid as described in the methods.-   Part C: A 200 mM solution of citric acid is adjusted to pH 4.5 with    200 mM sodium hydroxide. A cellulose based viscosity-enhancing agent    (hydroxypropylmethylcellulose, Methocel E4M from Dow Gmbh, Germany)    is added at 1% W/V: the polymer is sifted in while vigorously    stirring the sodium citrate solution and allowed to hydrate for 30    minutes.

A test preparation of PTC+Caprylic acid (PTC+Cap) was prepared by mixingequal amounts of Part A and Part C, yielding an emulsion of 0.125%caprylic acid with 0.1% PTC in 100 mM sodium citrate containing 0.5% W/VMethocel.

A test preparation of SGM+Caprylic acid (SGM+Cap) was prepared by mixingequal amounts of part B and Part C, yielding an emulsion of 0.125%caprylic acid with 0.1% SGM in 100 mM sodium citrate containing 0.5% W/VMethocel.

A test combination preparation comprising fast and slow acting emulsionswas prepared by mixing 30% PTC+Cap with 70% SGM+Cap and combining thiswith an equal volume of part C (30:70 blend), The 30:70 blend is anemulsion of 0.03% caprylic acid in 0.075% PTC combined with 0.07%caprylic acid in 0.0875% SGM in 100 mM sodium citrate containing 0.5%Methocel.

The microbicidal and adhesion inhibitory properties of all three testpreparations were assessed using the standard viability and adhesioninhibition assay described in the Methods. The inoculum for both assayswas an 18 hour broth culture of Candida albicans, grown in YEPD mediumand concentrated ×10 by centrifugation and re-suspension in one tenthvolume of supernatant.

12.0 gram samples of each test preparation were dispensed to Sterilintubes and each of these was inoculated with 1.2 ml aliquots ofconcentrated yeast culture. Samples of 1.0 ml volume were withdrawn formthese inoculated tubes at timed intervals over the course of one hourand added to 9.0 ml of diluting buffer containing 3% Tween 80 toneutralize. Serial dilutions and plate counting was undertaken toenumerate residual viability as described in the Methods. The resultsare presented in Table 15 and illustrated in FIG. 7.

TABLE 15 Mucosal Fortification: Viability of Candida albicans inTailored Release Preparations Minutes 0 5 10 15 20 25 30 35 40 45 50 55Blank 6.81 6.92 6.79 6.85 6.97 6.76 6.84 6.99 6.69 6.95 6.78 6.9 PTC +6.5 4.84 3.13 1.72 0 Cap SGM + 6.84 6.55 6.12 5.85 5.42 4.87 4.22 3.763.13 2.79 2.22 1.84 Cap 30:70 6.73 5.21 4.14 3.56 3.12 2.8 2.42 2.161.89 1.55 1.33 0.89 blend

The fast acting PTC+Cap behaved as expected reducing viability to zerodetectable cells in 20 minutes. The slow acting SGM+Cap was also asexpected with viability being reduced by 5 logs in 55 minutes. Thecombination 30:70 blend gives a good example of a ‘tailored release’profile, viability was reduced by more than 2 logs in 10 minutes, a ratewhich paralleled the fast acting PTC+Cap, thereafter the microbicidalrate slowed considerably, and approximated to that of the slow actingSGM+Cap. The blend however had achieved 1 log greater reduction inviability at 55 minutes compared to SGM+Cap alone.

Assessment of adhesion inhibitory properties of the three testpreparations was undertaken using the Buccal Epithelial Cell modeldescribed in the Methods and used in Example 1. For comparison,preparations of the two membrane lipids, PTC and SGM, at 0.1% in 100 mMsodium citrate buffer pH 4.5 with 0.5% Methocel were also prepared andincluded in the test procedure.

Washed yeast cell pellets were re-suspended in 2.5 ml volumes of thetest preparations (PTC, PTC+Cap, SGM, SGM+Cap, 30:70 blend describedabove and BSA blank). 100 mM sodium citrate at pH 4.5 was used fordetermination of control adhesion. After 10 minutes pre-exposure, theyeast suspensions were used to re-suspend washed Buccal Epithelial Cellpellets which had been harvested and prepared as described in theMethods. The combined yeast and Buccal Epithelial cell in test, blank orcontrol were incubated with gentle agitation for 60 minutes at 37° C.,after which direct microscopic counts using a Hemocytometer slide wereused to enumerate the numbers of yeast adhering to 100 Buccal Epithelialcells: The results are presented in Table 16 and illustrated in FIG. 8.

TABLE 16 Mucosal fortification: Inhibition of Adhesion in Tailoredrelease Preparations: Candida albicans to Buccal Epithelial CellCount/100 BEC % Adhesion % Inhibition Blank 509 100 0 PTC 239 47 53 SGM374 73 27 PTC + Cap 20 4 96 SGM + Cap 266 52 48 30:70 Blend 88 17 830.5% BSA 425 83 17

The adhesion inhibitory properties of 0.1% PTC on its own and with0.125% caprylic acid are considerably better than equivalentpreparations of SGM: as might be expected the 30:70 blend lies midwaybetween the two.

From the data in Table 16 it is also evident that the emulsified fattyacid has a synergistic effect on the adhesion inhibitory properties ofthe membrane lipid. The percentage reduction in adhesion achieved withPTC alone (53%), is reduced by a further 43% in combination withcaprylic acid.

It should be noted that according to the viability data in Table 16 andas illustrated in FIG. 7, none of the yeast cells in PTC+Cap is viableafter 20 minutes exposure, and approximately 50% to 60% of those in theother two samples are dead. Under the microscope however, yeast cellsappear to be intact and while greatly reduced there are still a fewapparent that are adhering to Buccal Epithelial cells, suggesting thatdead cells are capable of adhesion and biofilm formation.

Example 11: The Use of Membrane Lipids in Skin Antisepsis and Preventionof Cross Contamination in Hospitals and Patient Care Establishments

Methods of evaluating skin antiseptic agents in wash and gelformulations are well established and are fully described in theofficial procedures of the EU for ISO Certification under EN 1500 (handgel) and EN 1499 (liquid soap).

In this Example, the relatively non-pathogenic Escherichia coli K12 NCTC10538 was used (The National Collections of Industrial and MarineBacteria Ltd, UK: Catalogue of Type Strains ISBN No 0 9510269 3 3). Thebacterium is routinely cultured and maintained on tryptone soya agar orbroth (TSB), which may be acquired commercially from Oxoid, UK and hasthe following constituents: 1.5% W/W tryptone (pancreatic digest ofcasein), 0.5% W/W peptone (papaic digest of soybean meal), 0.5% W/Wsodium chloride, and agar at 1.5% W/W when required as a solid medium.

Prior to the test volunteers wash their hands with a mild non-antisepticsoap; a suitable product is E45 Emollient Wash Cream from BootsHealthcare, UK. After washing and drying, the hands are dipped into a 2litre beaker containing 1 litre of 24 hour culture of E. coli grown inTSB and containing not less than 2×10⁸ viable cells per ml as confirmedby serial dilution and plate counting. Both hands are immersed in thecontaminating suspension up to the mid-metacarpals and held there for 5seconds, and then removed. Excess contaminating fluid is allowed torun-off and then the hands are air dried in the horizontal position for3 minutes.

To ensure adequate contamination and to establish a pre-value forenumerating reduction, the hands are sampled by dipping the tips of thefingers and thumb of each hand into 10 ml of sterile PBS in aPetri-dish, and rubbed against the base of the plate for 1 minute. Aftersampling, the hands are treated with either the product of thisinvention or Spirigel; 4 ml of either preparation is applied to thehands and manipulated over the entire surface area of both hands. Thehands are then rinsed under clean (potable) running water, which islukewarm at approximately 37° C. for a timed period of 20 seconds. Afterrinsing, the hands are held in an upright position while an assistantdries the palms and wrists with a paper towel. The finger tips and thumbare then sampled by immersion in 10 ml of PBS as described above.

Immediately after sampling, prior to or after washing, 1 ml of thesampling fluid was aseptically transferred to and spread on the surfaceof a TSB agar plate and another 1 ml is transferred aseptically to 9 mlof sterile PBS and mixed and the process of serial dilution and platecounting proceeds as described previously.

A test preparation of the product of this invention was prepared, beinga combination of caprylic acid in phosphatidyl choline at 30% andcaprylic acid in sphingomyelin at 70% prepared as described in theMethods. In this Example the membrane lipids were prepared as 1%concentrates with 1% caprylic acid and blended at 30:70 ratio.

A viscosity-enhancing agent is employed to improve the rheology of thetest preparation. In this case a Carbopol co-polymer, Pemulen TR-1 fromNoveon Inc, Cleveland Ohio was used at 0.45%. The polymer was added to avolume of absolute ethanol equivalent to 70% of final preparation volumeand allowed to disperse therein for 30 minutes. A 30% volume of 30:70blend of the product of this invention as described above was then addedto the ethanol and polymer with constant stirring to facilitate rapiddispersion.

The final test product contains:

0.045% Caprylic acid in 0.045% Phosphatidylcholine: 0.105% Caprylic acidin 0.105% Sphingomyelin: 0.45% Carbopol polymer; 29.25% water; 70%ethanol.

Spirigel is reported to contain 70% ethanol, 30% water and an unknownamount of an unknown viscosity-enhancing agent.

In this Example both test product and Spirigel were used immediatelyafter experimental contamination of volunteers' hands to evaluatede-contamination, and both were used on experimentally re-contaminatedhands 10 minutes after application of Spirigel and test preparationaccording to the invention; the results are presented in Table 17.

TABLE 17 Persistent Effect of tailored release membrane lipids in skinantisepsis Spirigel Test item Pre-treatment contamination 6.42 6.69 Posttreatment contamination, i.e. 3.23 (−3.19) 3.42 (−3.27) residualviability Pre-application 10 min pre- Not Applicable Not Applicablecontamination (treatment 10 min pre-contamination) Contamination: 10 minpost 6.42 6.59 treatment Contamination: 10 min Post 5.97 2.67 (−3.3) application, i.e. viable cells recovered for contamination 10 mins postapplication

As might be anticipated from the alcohol content, when used immediatelyafter contamination both products achieved greater than 3 log reductionsof the applied contamination. When used on hands 10 minutes prior tocontamination however the alcohol content of both products hadevaporated by the time the contamination was applied, and the resultsshow that Spirigel had no significant residual effect (0.45 logreduction). The persistent nature of the membrane lipid emulsion offatty acid was still present on the hands in the test item, and achievedgreater than 3 log reduction. Spirigel has an immediate but nopersistent antimicrobial effect, while the test item according to thisinvention is both immediate and persistent microbicidal effects.

Example 12: Use of Membrane Lipid Emulsions in Wound Care

To illustrate the potential utility of the product of this invention inwound care an ex-vivo model employing freshly slaughtered sections ofbeef brisket is used. Brisket has an optimal distribution of leanmuscle, fat and collagen, and is consequently considered to berepresentative of all potentially infected wound surfaces. Brisketsections are excised immediately post slaughter, without chilling andwith the external fascia membrane intact, these are divided underaseptic conditions into cubes of approximately 1 cm square. Preparedcubes are ‘infected’ by submerging them in late log phase cultures ofbacteria for one minute, dried and suspended in a 37° C. environment forone hour to facilitate bacterial adherence and colonization of the meatsurfaces.

A suitable example of a wound care formulation is the “standardformulation” of this invention (containing 0.5% caprylic acid in 0.4%DLL) diluted 1:1 in 200 mM sodium citrate buffer at pH 4.5, which thenconsists of 0.25% caprylic acid in 0.2% DLL in 100 mM sodium citratebuffer at pH4.5

Test sections of infected meat are treated by spraying the wound careformulation directly onto the infected surface and evaluating the testsamples for residual bio-burden at predetermined exposure times.Infection and its eradication are confirmed by mechanical maceration oftreated and untreated sections in sterile phosphate buffered saline(PBS) and enumeration of the bio-burden by standard microbiologicalserial dilution and plate counting techniques.

Typical results are presented in FIG. 9, where greater than 7 logs ofthe common wound infecting bacteria, Staphylococcus aureus andStreptococcus pyogenes are shown to be eradicated in less than 120minutes. It should be appreciated that the rate of kill on a fissuredsurface such as a wound is extended due to the nature of the surface andthe time required for the formulation to permeate to the seat of theinfection.

A comparison of the relative potency of the standard formulation incitrate buffer as above with the alternative and commonly usedantimicrobials, chlorhexidine gluconate and silver sulphadiazine ispresented in FIG. 10. At 100 minutes exposure the product of thisinvention has virtually eradicated 7 logs of Staphylococcus aureus: 1log remains under chlorhexidine treatment and 3.5 logs with silversulphadiazine.

Example 13

The Surgical Use of Membrane Lipids in Prevention and Disruption ofBiofilm

Staphylococcus aureus RN4220 is noted for its ability to form tenaciousbiofilm under laboratory conditions when stress cultured in the presenceof sodium chloride. The organism was cultured to mid log phase (10hours) in Brain Heart Infusion broth and 20 micro-liter volumes used toinoculate wells of a 96 well microtitre plate containing 180 μl of BHIsupplemented with 4% sodium chloride. When incubated at 37° C. underthese conditions for 6 hours an appreciable biofilm is formed at thebase of each well. The biofilm is quantified by decanting the cultureand washing the wells with sterile distilled water, after which theplates are dried and stained with 0.4% crystal violet, re-washed anddried; the Optical Density of the stained biofilm is measured at 570 nm.

From previous Examples it will be clear that incorporation of themembrane lipid emulsified product of this invention will have amicrobicidal effect, preventing growth and biofilm formation. Asillustrated here where biofilm already exists, however, contact with anemulsion of membrane lipid and free fatty acid will effectively kill allviable bacteria in the biofilm and disrupt the film itself.

Assessment of reduction of viability of an established biofilm preparedas described above was achieved by incorporating Alamar Blue at 10% byvolume in fresh BHI broth and re-charging the wells of a 96 wellmicrotitre plate with pre-formed biofilm. Alamar Blue is a redoxindicator from Invitrogen Ltd, Paisley, UK. It imparts a deep blue colorto the media, and when reduced by microbial metabolic activity the colorchanges from non-fluorescent blue to a highly fluorescent red;absorbance and emerging fluorescence may be measured at 570 nm and 600nm.

Microtitre plate wells containing pre-formed biofilm were treated withthe membrane lipid CLS (ML CLS) used in Example 7 and similar wells werealso treated with Zuragen, Duralock and Taurosept for time periodsranging from zero to 60 minutes. At the end of each exposure period, theplates were decanted and washed once with phosphate buffered salinecontaining 3% Tween 80 and twice with sterile distilled water. 200 μl ofBHI containing 10% Alamar Blue was added to the wells and the platesincubated for 60 minutes. There was no detectable color change in any ofthe wells treated with the membrane lipid CLS of this inventionindicating complete eradication of viability within the biofilm in lessthan one hour. All of the wells treated with Zuragen, Duralock orTaurosept had changed from blue to red demonstrating little or noreduction in viability in the established biofilm.

The efficacy of the membrane lipid CLS of this invention, and thecomparable ineffectiveness of the other three formulations in reducingthe actual amount of pre-formed biofilm may be demonstrated usingsimilar procedures.

Microtitre plate wells containing pre-formed biofilm were treated withthe four formulations for time periods from zero to 60 minutes, decantedand washed as described previously. The treated plates were dried andstained with 0.4% crystal violet, dried and the intensity of stain beinga measure of residual biofilm was recorded at 570 nm. The results areillustrated in FIG. 11 wherein it is evident that although somereduction in biofilm was achieved with Zuragen, Duralock and Taurosept,it is inconsequential in comparison to the near total eradication ofbiofilm achieved with the membrane lipid CLS (ML CLS) of this invention.

Example 14

Comparative Adhesion Inhibitory Properties

De-lipidised lecithin is a more potent adhesion inhibitory substancecompared to milk serum apo-proteins in WO 03/018049, wherein theapo-proteins are generated by lipase hydrolysis of whey proteins. Foradhesion inhibitory comparison, a whey protein hydrolysate was preparedas described in WO 03/018049 using Carbelac 80 whey protein concentrate.A whey protein isolate (Provon 190 from Glanbia PLC) was also testedcontemporaneously. For full comparison purposes a formulation of 0.5%W/V caprylic acid in 0.4% W/V de-lipidised lecithin was prepared in 100mM sodium citrate pH 4.5 as described in the Methods and included in thetest sequence. All test items were dispersed in 100 mM sodium lactatebuffer at pH 4.5. The results are presented in Table 18 below.

TABLE 18 Candida albicans per 100 Buccal Epithelial Cells % inhibition 0mg/ml 2.5 mg/ml 5.0 mg/ml 10 mg/ml 15 mg/ml at 5 mg/ml Carbelac 80 483395 320 260 189 33 Provon 190 514 313 277 113 53 46 Lipase digest ofCarbelac 491 330 154 48 0 68 80 De-lipidised lecithin (DLL) 504 130 33 00 93 0.5% Caprylic in 0.4% 517 46 0 0 0 100 DLL

While de-lipidised lecithin on its own is significantly better than thethree dairy based preparation, the emulsified combination ofde-lipidised lecithin and caprylic acid is the most potent.

Example 15: Comparative Microbicidal Effect

The MIC by agar dilution technique is also used here to demonstrate thesuperior potency of the “standard formulation” described hereinaboveover the product disclosed in WO2009/072097 comprising a blend of freefatty acids emulsified in the whey protein isolate, Provon 190 fromGlanbia PLC. The product of WO2009/072097 contains 28% by weight of freefatty acid blend, while the standard formulation of this inventionscontains just 0.5% by weight. In order to make a suitable comparisonbetween the two products the product of WO2009/072097 was diluted by5/28 in sterile distilled water to obtain a dispersion comprising 5.0%free fatty acid which was then diluted further and used to prepare agarplates ranging from 1.0% free fatty acid to 0.5%, 0.4%, 0.3%, 0.2%,0.1%, 0.075%, 0.05% and 0.025% (of fatty acid content).

For further comparison an emulsion was constructed using 0.5% caprylicacid in 0.4% Provon 190, using the emulsification agent fromWO2009/072097 instead of de-lipidised lecithin. The results are shown inTable 19 below.

TABLE 19 Comparative MIC values Formulation of invention Product of 0.5%Caprylic containing 0.5% WO/2009/ in 0.4% caprylic acid in Test organism072097 Provon 190 0.4% DLL Staph aureus RN4220 >0.4% >0.7% >0.1% Staphepidermidis >0.4% >0.7% >0.1% NCTC 11047 Streppyogenes >0.4% >0.7% >0.075% NCTC 8198 Strepfaecalis >0.4% >0.7% >0.075% NCTC 12697 E. coli >1.0% >1.0% >0.4% ATCC11698 Salmonella typhimurium >0.8% >0.8% >0.3% NCTC 74Pseudomanas >1.0% >1.0% >0.5% aeruginosa ATCC 27853Pseudomanas >1.0% >1.0% >0.5% fluorescens NCTC 10038 Candida albicansC.I. >0.4% >0.7% >0.075% Candida glabrata >0.4% >0.7% >0.1% NCPF 8750

For the two Staphylococci, the two Streptococci and the Candida sp, themeasured MIC for the standard formulation is one quarter or less thanthat of the product of WO2009/072097, indicating a four times greaterpotency. In the case of the two Pseudomonas sp, E. coli and Salmonella,the measured MIC is one half to one third less than the product ofWO2009/072097 again clearly demonstrating significantly greater potency.The measured MIC's for the emulsion of caprylic acid in Provon 190 aresignificantly greater (significantly less potent) than the standardformulation of this invention.

Example 16

Comparative Microbicidal and Adhesion Inhibitory Effect of DifferentFree Fatty Acids in Both Emulsified and Free Form

Fatty acids in free form (not emulsified) have relatively littlemicrobicidal effect primarily because of their insolubility in aqueousmedium. Emulsification in membrane lipids as described herein expandsthe relative surface area of the fatty acid and facilitates itsdispersal in aqueous medium. The membrane lipid emulsification agentalso facilitates contact and transfer of the fatty acid to a microbialcell surface. As illustrated in previous Examples, variablelipophylicity between different membrane lipids affects the rate ofmicrobicidal effect. In general, membrane lipids are superioremulsification agents, exhibiting superior microbicidal effect asdemonstrated in Example 15. As demonstrated here the superior potency ofmembrane lipid emulsified free fatty acid extends across a range ofmicrobicidal fatty acids.

Seven separate emulsions of seven different free fatty acids at 0.5% W/Vwere prepared in 0.4% W/V de-lipidised lecithin as described in themethods. Emulsions were prepared at temperatures above the meltingpoints of the individual free fatty acids: caproic, caprylic andpelargonic at room temperature (20° C.); capric and undecylenic acidwere prepared at 37° C., lauric acid was emulsified at 50° C. andmyristic at 60° C. A blend of 50% caproic and 50% lauric acid has amelting point of less than 28° C. as does a blend of 40% lauric acid inoil of lemon balm, and these were emulsified at 37° C.

The comparative microbicidal effect of each individual fatty acid andblend thereof in free non-emulsified form and emulsified in de-lipidisedlecithin was evaluated using the microbicidal suspension test describedin the methods. Evaluation of the microbicidal effect of freenon-emulsified fatty acids is frustrated by their insolubility.Inclusion of the non-emulsified form was considered necessary toillustrate the exponential increase in potency in the emulsified form.Each free fatty acid was prepared at 0.5% W/V in water and agitatedvigorously to disperse followed by immediate pipetting of 1.0 mlaliquots to test containers, prior to inoculation with the testorganism. The test organism was Staphylococcus aureus RN 4220 grown tolate log phase on Brain Heart Infusion Broth.

The results are presented in Table 20 below.

TABLE 20 Comparative Microbicidal Effect Free fatty acids or blendsthereof at 0.5% W/V non-emulsified in the test and at 0.5% W/Vemulsified in 0.4% W/V de-lipidised lecithin in the test. Exposure Timein Seconds at 37° C. Fatty acid or blend 0 60 120 180 240 300 360 Freecaproic acid 7.87 7.17 6.82 7.57 6.93 6.47 6.73 Emulsified caproic 7.876.21 4.89 2.53 0 0 0 acid Free caprylic acid 6.94 6.79 6.83 6.21 6.335.89 6.17 Emulsified caprylic 6.94 5.32 3.71 0 0 0 0 acid Freepelargonic acid 6.94 6.55 6.31 5.96 5.67 5.83 5.27 Emulsified pelargonic6.94 4.63 2.99 1.81 0 0 0 acid Free capric acid 6.94 6.32 6.48 6.73 6.365.97 6.11 Emulsified capric acid 6.94 4.79 3.44 2.19 0 Free undecylenicacid 7.87 7.19 6.99 6.57 6.83 6.67 6.31 Emulsified 7.87 4.97 2.69 0 0 00 undecylenic Free lauric acid 7.87 7.35 7.77 7.41 7.98 7.23 7.65Emulsified lauric acid 7.87 5.79 4.63 3.86 2.55 0 0 Free myristic acid7.38 7.27 7.84 6.93 7.11 6.26 6.84 Emulsified myristic 7.38 6.46 5.815.21 4.98 3.69 2.99 Free lauric caproic 7.38 6.99 7.18 6.74 6.37 6.956.81 50:50 Emulsified Lauric: 7.38 6.76 5.32 3.17 2.98 0 0 caproic Free40% lauric in oil 7.38 7.21 7.76 7.39 6.95 6.87 6.49 of lemon balmEmulsified 40% Lauric 7.38 5.92 3.13 2.77 0 0 0 in Oil of Lemon BalmDe-lipidised Lecithin 7.33 7.92 7.41 6.83 7.14 7.66 6.95 0.4% W/V Blankdeterminations were conducted in all assays by suspending the inoculumin phosphate buffered saline at 37° C. and no reduction in inoculumviability was detected over the six minute exposure period in any testsequence.

Free fatty acids in non-emulsified form achieve at best 1 log reductionin viability over the test period of 6 minutes. The membrane lipidemulsification agent (de-lipidised lecithin) equally exerts little or nomicrobicidal effect. In comparison, with the exception of emulsifiedmyristic acid, equivalent weight emulsions of all other fatty acids andblends thereof in de-lipidised lecithin reduce residual viability in atest inoculum of 6.94 to 7.87 logs to zero in less than 4 minutes.

Neither free fatty acids on their own nor the de-lipidised membranelipid on its own have any detectable microbicidal effect within thetime-frame of this test. An emulsified combination of the two enablesthe microbicidal effect of the fatty acid synergistically.

The same test items used in microbicidal evaluation above were alsotested for their adhesion inhibitory properties using the BuccalEpithelial Cell assay described in the Methods.

TABLE 21 Comparative Adhesion Inhibitory Effect Free fatty acids orblends thereof at 0.5% W/V non-emulsified in the test and at 0.5% W/Vemulsified in 0.4% W/V de-lipidised lecithin in the test. Blank Test % %Count/ Count/ Adhe- inhi- Fatty acid or blend 100 BEC 100 BEC sionbition Free caproic acid 429 380 89 11 Emulsified caproic acid 503 0 0100 Free caprylic acid 429 400 93 7 Emulsified caprylic acid 503 0 0 100Free pelargonic acid 429 360 84 16 Emulsified pelargonic acid 503 2 0.399.7 Free capric acid 429 386 90 10 Emulsified capric acid 503 0 0 100Free undecylenic acid 521 448 86 14 Emulsified undecylenic 508 0 0 100Free lauric acid 474 450 95 5 Emulsified lauric acid 508 56 11 89 Freemyristic acid 474 436 92 8 Emulsified myristic 508 68 13 87 Free lauriccaproic 50:50 474 450 95 5 Emulsified Lauric: caproic 508 0 0 100 Free40% lauric in oil of 526 460 87 13 lemon balm Emulsified 40% Lauric inOil 526 0 0 100 of Lemon Balm De-lipidised Lecithin 0.4% 497 96 19 81W/V

De-lipidised lecithin on its own in the test achieves 81% inhibition ofadhesion. Emulsions of caproic, caprylic, pelargonic, capric andundecylenic achieve greater than 99% inhibition, and emulsions of lauricand myristic achieve greater than 86% inhibition. All non-emulsifiedfree fatty acids achieve less than 17% inhibition. The adhesioninhibitory effect of caprylic acid for example is amplified by a factorof 14 in emulsified form.

At the concentrations of test item used in Table 21, the adhesioninhibitory effect is essentially swamped by the intrinsic microbicidaleffect. It has been demonstrated that the majority of microbial cellswill be dead as a result of exposure to the microbicidal effect of theemulsified fatty acid, and it must be assumed that this will influenceadhesion.

A more appropriate measure of the amplified adhesion inhibitory effectattributable to emulsions vs free acid or non-emulsified membrane lipidsmay be obtained using a concentration below the Minimum InhibitoryConcentration (MIC) of the emulsified free fatty acid. The MIC ofcaprylic acid in the formulation of this invention is greater than 0.1%.

A formulation of 0.5% caprylic in 0.4% de-lipidised lecithin (as usedabove) is diluted by a factor of 5 in sterile distilled water to achievea concentration of 0.1% caprylic in 0.08% de-lipidised lecithin andfurther diluted by half to achieve 0.05% caprylic in 0.04% DLL. Thesedilutions together with the same concentrations of DLL andnon-emulsified free caprylic acid were tested in the Buccal EpithelialCell assay as above. The results are presented in Table 22.

TABLE 22 Low Dose Adhesion Inhibitory Effect Caprylic acid at 0.1% W/Vand 0.05% W/V non-emulsified in the test and at 0.1% W/V and 0.05% W/Vemulsified in 0.08% W/V and 0.04%% W/V DLL respectively in the test,together with 0.08% and 0.04% non-emulsified DLL. Blank Test % % Count/Count/ Adhe- inhi- 100 BEC 100 BEC sion bition Free caprylic acid 0.1%489 466 95 5 Free caprylic acid 0.05% 487 459 94 6 DLL 0.08% 533 421 8515 DLL 0.04% 509 453 87 13 0.1% caprylic in 0.08% DLL 499 255 51 490.05% caprylic in 0.04% DLL 513 303 59 41

As illustrated in Table 22, the addition of caprylic acid atconcentrations below its MIC (0.1% and 0.05%) amplifies the adhesioninhibitory effect of 0.08% DLL and 0.04% DLL by more than a factor ofthree in both cases.

Example 17: Reduction of Antagonistic Effect on Mammalian Cells

Mammalian cell membranes are susceptible to disruption by free fattyacids in a manner not dissimilar to their effect on microbial cellmembranes. The effect is not classical cell toxicity as it relates tosuperficial cell surface damage and not interference with metabolicprocess or nucleic acid replication. It is ameliorated significantly bybody fluids in vivo. Protection against mammalian cell membrane damagecan be enhanced by adding additional amounts of free membrane lipid to amembrane lipid emulsion of a free fatty acid. The protective effect isnot confined to the use of membrane lipids. As illustrated here, milkserum whey protein isolate also serves as a suitable, although notoptimal barrier against mammalian cell damage.

The test item is an emulsion of 0.5% caprylic acid in 0.4% de-lipidisedlecithin prepared as described in the Methods, and combined with anequal volume of 200 mM sodium citrate at pH 4.5 also prepared asdescribed in the Methods. Dispersions of 0.4% and 0.8% de-lipidisedlecithin were prepared in 200 mM sodium citrate at pH 4.5, and combinedin equal volumes with aliquots of the emulsion of caprylic acid inde-lipidised lecithin to achieve emulsions of 0.25% caprylic acid in0.2% de-lipidised lecithin suspended in an aqueous solution of 100 mMsodium citrate at pH 4.5 in which further amounts of either 0.2% or 0.4%de-lipidised lecithin were dispersed, these are described as Test+0.2%DLL or Test+0.4% DLL

Similar suspensions of the same emulsion were prepared in sodium citratedispersions of a Whey Protein Isolate (WPI) (Provon 190 from GlanbiaPLC) and with Bovine Serum Albumin for comparison purposes. These aredescribed as Test+0.2% or Test+0.4% WPI or BSA.

Raji B lymphocytes were grown as described in the Methods and viabilityafter a 60 minute exposure to the various test solutions was assessedusing an Invitrogen Countess Cell Viability meter as described in theMethods. The results are presented in Table 23 below.

TABLE 23 Reduction in cell viability at 60 minute exposure to test itemsRaji B Lymphocytes Test + Test + Test + Test + Test + Test + Buffer 0.2%0.4% 0.2% 0.4% 0.2% 0.4% control blank Test DLL DLL WPI WPI BSA BSA % 7872 75 77 73 76 79 75 71 Viable T zero % 72 69 12 59 68 48 53 19 21Viable T 60 min % cell 92 96 16 77 93 63 67 25 30 survival

The % cell survival in the test item is just 16% after 60 minutes. Bycomparison, the control consisting of Raji B cells in cell culture medialost just 8% viability and the buffer blank being 100 mM sodium citrateat pH 4.5 was even less at 4% reduction. With the addition of 0.2% and0.4% free membrane lipid (de-lipidised lecithin) % cell survival in thetest was increased from 16% to 77% and 93%, and although not quite aseffective, the addition of free WPI increased cell survival from 16% to63% and 67%. Bovine serum albumin was considerably less effective inprotecting against cell damage.

The inclusion of free membrane lipid in an aqueous dispersion ofmembrane lipid emulsion has no significant effect on the microbicidalproperties of the membrane lipid emulsion. A late log phase culture ofthe yeast Candida albicans grown as described in the Methods contained1.33×10⁷ viable cells per ml. This was used to inoculate aliquots ofeach test item from above at a dilution of 1:10 such that each ml oftest item contained in excess of 6 logs of yeast cells. Samples werewithdrawn over time periods of up to 10 minutes and assessed forresidual viability using the procedures described in the Methods. Asillustrated in Table 24 below, detectable viability was eradicated inless than 5 minutes by all test items.

TABLE 24 Microbicidal Effect of Free Membrane Lipid in Membrane LipidEmulsions Time to kill greater than 6 logs Candida albicans. Test +Test + Test + Test + Test + Test + Buffer 0.2% 0.4% 0.2% 0.4% 0.2% 0.4%control blank Test DLL DLL WPI WPI BSA BSA Time NA NA <5 <5 <5 <5<5 >5 >5 Mins

Example 18: Use in a Medical Food

Separate emulsions of caprylic, capric and lauric acid were prepared as5.0% W/V fatty acid emulsified in 4.0% de-lipidised lecithin asdescribed in the Methods. The individual emulsions were mixed in a ratioof 1:1:1.

Marvel skim milk powder from Premier International Foods (UK) Ltd,Spalding, Lincolnshire, England was re-constituted using 90% of thewater volume according to the manufacturer's instructions. Once fullyhydrated 10% by volume of the combined mix of separately emulsified freefatty acids was added and mixed by stirring, bringing the total volumeto 100%.

Helicobacter pylori was grown on Columbia Blood Agar supplemented with5% de-fibrinated sheep blood in an anaerobic jar using Anaerogen lowoxygen gas packs from Oxoid UK. Salmonella typhimurium and E. coli K12were grown on Brain Heart Infusion agar as described in the Methods.

The microbicidal efficacy of the re-constituted milk supplemented withthe three individually emulsified free fatty acids was determined usingthe Minimum Inhibitory Concentration (MIC) method of agar dilutiondescribed in the methods. Dilutions of the re-constituted skim milk wereprepared in sterile distilled water such that further dilutions ofaliquots of these in cooled agar provided an agar with combined freefatty acid concentrations ranging from 1% to 0.1% in 0.1% increments andfrom 0.1% to 0.01% in increments of 0.01%. Cultures of the three testorganisms were inoculated onto these plates and incubated according toculture requirements: Helicobacter under low oxygen tension, Salmonellaand E. coli under aerobic conditions all at 37° C.

The minimum Inhibitory Concentration, being the lowest dilution where nogrowth was observed was greater than 0.5% for Helicobacter and greaterthan 0.1% for both Salmonella and E. coli.

The invention claimed is:
 1. A method of treating or preventing aninfection in a subject in need thereof, the method comprisingadministering to the subject an effective amount of a topicalantimicrobial composition which inhibits microbial adhesion, wherein thetopical antimicrobial composition comprises: (a) a saturated orunsaturated free fatty acid selected from caprylic, capric, lauric andundecylenic acids and mixtures thereof or a pharmaceutically acceptablesalt thereof; and (b) delipidised lecithin containing less than 3%conjugated extraneous lipid material; and wherein the ratio of component(a) to component (b) is from 0.25:1 to 10:1; and wherein the infectionis an infection of the skin, nails, hair or mucosal surfaces of the eye,nose, inner ear, throat, lungs, gastro-intestinal tract, urinary tractor genitalia.
 2. The method of claim 1, wherein the free fatty acid isselected from caprylic and capric acids.
 3. The method of claim 2,wherein the free fatty acid is caprylic acid.
 4. The method of claim 1,wherein the ratio of component (a) to component (b) in the compositionis from 0.5:1 to 10:1, 0.5:1 to 5.0:1, 1.0:1 to 2.5:1, or 1.25:1 to2.5:1 on a weight for weight basis.
 5. The method of claim 1, whereinthe infection is an infection of the mucosal surfaces of thegastro-intestinal tract, urinary tract or genitalia.
 6. The method ofclaim 1, wherein the composition is a liquid soap, gel, solution,ointment, spray, mouthwash, bandage, wound dressing, saliva supplementor enema.
 7. The method of claim 1, wherein the infection is aninfection of the skin.
 8. The method of claim 7, wherein the infectionis caused by gram positive bacteria, gram negative bacteria, yeast,fungi, Staphylococcus aureus, Streptococus pyogenes, Pseudomonas,Propionibacteriium, Trichophyton, Microsporum, Epidermophyton, Candidaalbicans, Malassezia furfur, Enveloped viruses, Methicilin ResistantStaphylococcus aureus (MRSA), Vancomycin Resistant Enterococci (VRE), orClostridium difficile.
 9. The method of claim 8, wherein the Envelopedviruses is Herpes Simplex Type 1, Herpes Simplex Type 2, Herpes zoster,Molluscum, HIV, SARS, Swine flu, bird flu, or Hepatitis.
 10. The methodof claim 8, wherein the infection results in acne, Impetigo,Folliculitis, Erysipilis, Cellulitis, Necrotising Fasciitis or Tinnea.11. The method of claim 7, wherein the composition is liquid soap, gelor ointment.
 12. The method of claim 5, wherein the infection is aninfection of the musosal surfaces of the mouth, esophagus, stomach,large intestine, small intestine, rectum, vagina, external labia, or theglans and the lining of the urinary tract.
 13. The method of claim 1,wherein the infection is an infection of the mucosal surfaces of theinner ear, nose, naso-pharyngeal surfaces, throat, trachea, lungs orbronchioli.
 14. The method of claim 12, wherein the infection is aninfection of the mucosal surfaces of the mouth.
 15. The method of claim14, wherein the infection is gum disease, aphthous ulcer, halitosis orxerostomia.
 16. A method of treating or preventing an infection on thesurface of a tooth of a subject in need thereof, the method comprisingadministering to the subject an effective amount of an antimicrobialcomposition which inhibits microbial adhesion, wherein the antimicrobialcomposition comprises: (a) a saturated or unsaturated free fatty acidselected from caprylic, capric, lauric and undecylenic acids andmixtures thereof or a pharmaceutically acceptable salt thereof; and (b)delipidised lecithin containing less than 3% conjugated extraneous lipidmaterial; and wherein the ratio of component (a) to component (b) isfrom 0.25:1 to 10:1.
 17. The method of claim 16, wherein the infectionis dental caries.
 18. The method of claim 16, wherein the composition isa toothpaste, dry residue, liquid, cream, gel, paste, ointment, powder,spray or fibrous wipe.
 19. A method of inhibiting microbial adhesion onthe skin, nails, hair or mucosal surfaces of the eye, nose, inner ear,throat, lungs, gastro-intestinal tract, urinary tract or genitalia of asubject having or at risk of having a microbial infection, the methodcomprising administering to the subject an effective amount of anantimicrobial composition wherein the antimicrobial compositioncomprises: (a) a saturated or unsaturated free fatty acid selected fromcaprylic, capric, lauric and undecylenic acids and mixtures thereof or apharmaceutically acceptable salt thereof; and (b) delipidised lecithincontaining less than 3% conjugated extraneous lipid material; whereinthe ratio of component (a) to component (b) is from 0.25:1 to 10:1. 20.A method of treating or preventing an infection in a subject in needthereof, the method comprising administering to the subject an effectiveamount of an antimicrobial composition which inhibits microbialadhesion, wherein the antimicrobial composition comprises: (a) asaturated or unsaturated free fatty acid selected from caprylic, capric,lauric and undecylenic acids and mixtures thereof or a pharmaceuticallyacceptable salt thereof; and (b) delipidised lecithin containing lessthan 3% conjugated extraneous lipid material; and wherein the ratio ofcomponent (a) to component (b) is from 0.25:1 to 10:1; and wherein theinfection is an infection of the skin, nails, hair or mucosal surfacesof the eye, nose, inner ear, throat, lungs, gastro-intestinal tract,urinary tract or genitalia.